Methods and compositions for the treatment and prevention of hypoglycemia and related disorders

ABSTRACT

A composition comprising chromium and insulin and/or a chromium-insulin complex, its method of preparation, and its use in the prevention and treatment of hypoglycemia and hypoglycemia-related conditions. This composition can be administered in numerous ways, including parenterally, intranasally, and orally. The composition stabilizes serum glucose levels and has a synergistic effect compared to chromium and insulin administered separately.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/409,960, filed Mar. 1, 2012, which claims the benefit of U.S.Provisional Patent Application No. 61/448,134, Mar. 1 2011, all of whichare incorporated herein by reference in their entirety, including anydrawings.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The embodiments disclosed herein relate to compositions for thetreatment and prevention of hypoglycemia and hypoglycemia-relatedconditions, e.g., arising from insulin administration, and methods ofmaking and using the same. Also provided are improved methods ofadministering insulin and treating diabetes.

2. Background of the Invention

Glucose-metabolism Related Diseases and Disorders

Many diseases and disorders have been associated-etiologically orotherwise—to impaired, altered, or abnormal glucose metabolism. Thesediseases and disorders include, but are not limited to: diabetes(hyperglycemia); hypoglycemia; cardiometabolic syndrome; Alzheimer'sdisease; Huntington's disease; epilepsy; ischemia; Parkinson's disease;amnesia; dementia; mild cognitive impairment (MCI); attention deficithyperactivity disorder (ADHD); amyotrophic lateral sclerosis (ALS); and,traumatic brain injury.

Hypoglycemia

Hypoglycemia is a term that literally means “low blood sugar.”Hypoglycemia includes a state of a blood glucose level of not higherthan about 60 mg/dL, but is not limited to this blood glucose level. Forexample, when a person having high blood glucose due to diabetes or thelike undergoes a reduction in blood glucose level upon insulin injectionor the administration of an antidiabetic agent, or when a healthyindividual undergoes rapid reduction in blood glucose level due tohunger or strenuous exercise, similar conditions to hypoglycemia canappear even at about 100 mg/dL. Hypoglycemia often arises as a sideeffect of diabetes treatment (e.g., administration of insulin).Hypoglycemia can also result, however, from other medications ordiseases, hormone or enzyme deficiencies, or tumors. Furthermore,hypoglycemia can result from a long-term habit of ingesting largeamounts of carbohydrates; from excessive ingestion of alcohol; and fromcontinuation of extreme exercise for a long time in a state of dietaryinsufficiency. Hypoglycemia induced by diabetes treatment or othermedications are particularly dangerous, however, resulting in a higherprobability of a severe condition as compared to other causes ofhypoglycemia.

Hypoglycemia-related disorders and hypoglycemia-related complicationsrefer to conditions or complications that arise as a result of low bloodsugar, such as insulin-induced brain tissue damage, and the like.Hypoglycemia-related disorders and hypoglycemia-related conditions mayoccur where a reduction in glucose level in blood is accompanied by areduction in glucose level in the brain thereby causing lassitude,general discomfort, dismay, malaise, jitteriness, trembling, headache,weakness, cold sweat and palpitation, additionally causing impairedconsciousness and coma, which may also lead to death in a serious case.

Diabetes Mellitus

Diabetes mellitus is known to affect at least 10 million Americans, andmillions more may unknowingly have the disease. Diabetes is the sixthleading cause of death in the United States and accounted for more than193,000 deaths in 1997. Diabetes is a disease state in which thepancreas does not release insulin at levels capable of controllingglucose levels. Diabetes is classified into two types. The first type isdiabetes (Type 1) that is insulin dependent and usually appears in youngpeople. The islet cells of the pancreas stop producing insulin mainlydue to autoimmune destruction. Standard therapy for Type 1 diabetes isthe administration of insulin. Type 1 diabetic patients are the minorityof total diabetic patients (up to 10% of the entire diabeticpopulation). The second type of diabetes (Type 2) is non-insulindependent diabetes, which is caused by a combination of insulinresistance and insufficient insulin secretion. This is the most commontype of diabetes in the Western world. Close to 8% of the adultpopulation of various countries around the world, including the UnitedStates, have Type 2 diabetes, and about 30% of these patients will needto use insulin at some point during their life span due to secondarypancreas exhaustion.

The American Diabetes Association (ADA), World Health Organization (WHO)and Japan Diabetes Society (JDS) recently announced new diagnosticcriteria for diabetes, taking into consideration the achievements ofclinical and epidemiologic studies. Under these criteria, one isclassified as diabetic when any of the following blood glucose levelsare observed: fasting blood glucose ≧126 mg/dL; casual blood glucose≧200 mg/dL; or blood glucose two hours after the 75 g oral glucosetolerance test (OGTT) ≧200 mg/dL (Diabetes Care 20: 1183 (1997); DiabetMed 15: 539 (1998); and Diabetes 42: 385 (1999)).

Type 1 diabetics and many Type 2 diabetics, must manage their bloodglucose concentration with administration of insulin multiple times aday because their pancreas is not capable of producing adequate insulinwhich is necessary to support glucose metabolism. The goal ofadministrating the proper insulin dose is to maintain blood glucoseconcentrations close to the physiological norm, which is approximately 1gram of glucose per liter of blood, or 100 mg/dL. If not enough insulinis administered, the blood glucose level can reach hyperglycemic levels,leading to adverse health complications. Conversely, if too much insulinis administered, glucose levels can fall significantly below normal,creating a serious acute condition called hypoglycemia. It is a problemfor a diabetic patient to know his of her immediate requirement forinsulin, and it is not uncommon for diabetic patients to be off a factorof 2 or 3 from the desirable euglycemic target of 100 mg/dL. Poorlymanaged, the subject's blood glucose can alternate from hyperglycemic tohypoglycemic, or vice versa, in less than an hour. Hypoglycemia, if leftuntreated, can lead to seizures, brain damage, coma, or death. Thus,there is a need for improved methods of managing blood glucose levelswith insulin.

Brain Glucose Metabolism/Transporters and Associated Diseases andDisorders

Glucose homeostasis is critical for energy generation, neuronalmaintenance, neurogenesis, neurotransmitter regulation, cell survivaland synaptic plasticity. Glucose is the principle energy source formammalian brain, and a key role in cognitive function.

Delivery of glucose from the blood to the brain requires its transportacross the endothelial cells of the blood-brain barrier and across theplasma membranes of neurons and glia, which is mediated by thefacilitative glucose transporter proteins. Facilitative glucosetransport is mediated by one or more members of the closely-relatedglucose transporter (GLUT) family. Thirteen members of the GLUT familyhave been described thus far. Tissue-specific glucose transportersallocate glucose among organs in order to maintain brain glucoseconcentrations. The two primary glucose transporter isoforms whichfunction in cerebral glucose metabolism are GLUT-1 and GLUT-3. GLUT-1 isthe primary transporter in the blood-brain barrier, choroid plexus,ependyma, and glia; GLUT-3 is the neuronal glucose transporter. GLUT-4,on the other hand, carries glucose across the membranes of muscle andfat cells.

Insulin, a regulator of glucose uptake, is secreted by the pancreas.Insulin allocates glucose to muscle and fat. Thehypothalamus-pituitary-adrenal (HPA) axis, the sympathetic nervoussystem (SNS), and vascular endothelial growth factor allocate glucose tothe brain. Feedback pathways both from the brain and from muscle and fatare involved in regulating glucose allocation and exogenous glucosesupply. Further, insulin can cross the blood-brain barrier (BBB),reaching neurons and glial cells, and can exert a region-specific effecton glucose metabolism. Increased glucose consumption causes an increasein the net transport of glucose from blood to brain. It has been shownthat insulin-induced hypoglycemia increases brain GLUT-1 & GLUT-3levels. (Uehara et al. (1997) Am. J. Physiol. 272:E716-E719). Thus,insulin indirectly affects the transport without acting on the transportmechanisms. It has been proposed that part of the insulin action maytake place in extracerebral tissues via changes of the amino acidbalance in the blood. (Reagan et al. (1999) Am. J. Physiol. Endocrinol.Metab. 276:E879-E886).

GLUT-1 facilitates transport of glucose across the blood-brain-barrier.GLUT-1 expression levels are insulin-independent. Rather, GLUT-1 isdependent on potent regulators of blood vessel function like vascularendothelial growth factor (VEGF), a pituitary counter regulatoryhormone. HPA-axis overdrive causes metabolic abnormalities such ascentral adiposity, hyperglycemia, dyslipidemia, and hypertension, thatare well known clinical aspects the metabolic syndrome. Overexpressionof GLUT-1 in skeletal muscle is associated with marked increases inlactate and glycogen due to an increase in basal glucose uptake, andincreased glucose flux results in resistance of GLUT-4 to activation byinsulin and other stimuli, such as hypoxia and contractile activity(Katsumata et al. (1999) FASEB J. 11:1405-13).

GLUT-3, the neuron-specific glucose transporter, is solely responsiblefor the delivery of glucose into neurons in the central nervous system.GLUT-3 mRNA is widely expressed in the brain, including the pyramidalneurons of the hippocampus, the granule neurons of the dentate gyms, andthe cortex.

Brain-specific kinases 1 and 2 (BRSK1/2) are AMP-activated proteinkinase (AMPK)-related kinases that are highly expressed in mammalianforebrain. The activation of AMPK plays an important, albeit not anexclusive, role in the induction of recruitment of the insulin-dependentglucose transporter found in skeletal muscle, GLUT-4, to the plasmamembrane. The ability of AMPK to stimulate GLUT-4 translocation to theplasma membrane in skeletal muscle occurs via a mechanism distinct fromthat stimulated by insulin since together insulin and AMPK effects areadditive. In addition to its role in the regulation of GLUT-4, datasuggest that AMPK regulates glucose transport through GLUT-1.

Altered glucose metabolism in the brain is associated with variousdisease states, including but not limited to Alzheimer's disease,Huntington's disease, epilepsy, ischemia, amnesia, and traumatic braininjury. Glucose transporter expression is believed to be related toaltered glucose metabolism. Chronic hyperglycemia downregulates GLUT-1and GLUT-3 expression at both mRNA and protein levels in the brain,which is not due to the decrease of the density of microvessels. (Hou etal. (2007) Chin Med J (Engl). 120(19):1704-1709). The downregulation ofGLUT-1 and GLUT-3 expression might be the adaptive reaction of the bodyto prevent excessive glucose entering the cell that may lead to celldamage. Studies suggest that chronic stress produces molecular,morphological, and ultrastructural changes in the hippocampus that areaccompanied by cognitive deficits. Further, in insulin resistance,dementia, and cognitive impairment, and Alzheimer's disease, there is areduced sensitivity to insulin resulting in hyperinsulinemia. Toxiclevels of insulin negatively influence neuronal function and survival,and elevation of peripheral insulin concentration acutely increases itscerebrospinal fluid (CSF) concentration. Peripheral hyperinsulinemiacorrelates with an abnormal removal of the amyloid beta peptide (Abeta)and an increase of tau hyperphosphorylation as a result of augmentedcdk5 and GSK3beta activities. This leads to cellular cascades thattrigger a neurodegenerative phenotype and decline in cognitive function.

In Alzheimer's disease, glucose metabolism is decreased and isassociated with decreased amounts of GLUT-1 protein in cerebralmicrovessels in the frontal cortex and hippocampus, the regions mostaffected. (Kalaria et al. (1989) J. Neurochem. 53:1083-1088). Likewise,GLUT-3 levels have been reported to be reduced in the brains of patientswith Alzheimer's Disease. (Simpson et al. (1994) Ann. Neurol.35:546-551).

Studies have suggested that a condition termed mild cognitive impairment(MCI) represents prodromal Alzheimer's disease and if diagnosed earlyrepresents the best opportunity for pharmaceutical intervention. Theclinical criteria used for diagnosis of MCI are those of Petersen et al.(Arch Neurol (1999) 56:303-308) and include: memory complaintscorroborated by an informant; objective memory impairment for age andeducation; normal general cognitive function; intact activities of dailyliving; and, the subject does not meet criteria for dementia.

Huntington's disease is a neurodegenerative disorder. Early stages ofthe disease are characterized by subtle changes in personality,cognition, or physical skills. The most characteristic initial physicalsymptoms is chorea, characterized by jerky, random, and uncontrollablemovements. Chorea is often initially exhibited as general restlessness,small unintentionally initiated or uncompleted motions, uncoordination,or slowed saccadic eye movements. Symptoms such as rigidity, repetitivemotions or abnormal posturing appear as the disorder progresses. Thesesymptoms are regarded as the onset stage of the disease, and graduallybecome the dominant physical symptoms. Juvenile Huntington's Diseasediffers from these symptoms, in that it generally progresses faster andchorea is exhibited briefly, if at all, with rigidity being the dominantsymptom. Additionally, seizures are a common symptom of JuvenileHuntington's Disease. In Huntington's disease, GLUT-1 and GLUT-3 levelsare decreased in the caudate portion of the brain. (Gamberino et al.(1994) J. Neurochem. 63:1392-1397). Decreases in caudate glucosemetabolism have been reported in subjects with both symptomatic andclinically asymptomatic subjects at risk for Huntington's Disease.(Mazziotta, et al. (1987) New England J. Med. 316:357-362).

Glucose transport is also decreased in the human epileptic brain, due atleast in part to decreased expression of GLUT-1 at the blood brainbarrier endothelium (Cornford, et al. (1998) Ann. Neurol. 43:801-808;Cornford et al. (1998) J. Neuropathol. Exp. Neurol. 54:842-851).

Idiopathic epilepsy has a greater incidence amongst the Type 1 diabeticpopulation than the greater population (Hannonen et al. (2003)Developmental Medicine & Child Neurology 45:4:262-268). Meaningsinferred from the results could be interpreted in several ways. Diabetescould be partly responsible for idiopathic generalized epilepsy, or thetwo conditions could have different ages of onset. Metabolicabnormalities including hyperglycemia, mild hyperosmolality andhyponatremia contribute to the development of epilepsiapartialiscontinua in an area of focal brain damage. Occipital seizures andhemianopsia can be caused by hyperglycemia and may be accompanied byspecial MRI and VEP findings. The increased incidence of seizure anddelayed neuronal damage resulting from pre-ischemic hyperglycemiacorresponds with corticosterone levels rather than with glucose levelsand suggests that corticosterone has a greater prognostic value thanglucose in predicting cerebral ischemic damage.

GLUT-1 deficiency syndrome is a disorder that primarily affects thebrain. Affected individuals generally have seizures beginning in thefirst few months of life. Infants with GLUT-1 deficiency syndrome have anormal head size at birth, but growth of the brain and skull is oftenslow, in severe cases resulting in an abnormally small head size(microcephaly). Subjects with GLUT-1 deficiency syndrome often exhibitdevelopmental delay or intellectual disability. GLUT-1 deficiencysyndrome is also associated with other neurological problems, such asstiffness caused by abnormal tensing of the muscles (spasticity),difficulty in coordinating movements (ataxia), and speech difficulties(dysarthria). Some experience episodes of confusion, lack of energy(lethargy), headaches, muscle twitches (myoclonus), or involuntaryirregular eye movements, particularly before meals.

Other markers associated with brain glucose metabolism andtransport-related diseases and disorders include Nrf2 (nuclear factorerythroid 2 related factor 2), GFAP (glial fibrillary acidic protein),and HNE (4-Hydroxynonenal).

Nrf2 (nuclear factor erythroid 2 related factor 2) is a regulator ofmultiple cytoprotective proteins. Nrf2 is a transcription factor thatpositively regulates a transcriptional program that maintains cellularredox homeostasis and protects cells from oxidative insult (Rangasamy etal. (2004) J Clin Invest 114:1248). Nrf2 activates transcription of itstarget genes through binding specifically to the antioxidant-responseelement (ARE) found in those gene promoters. Decreased levels of Nrf2have been associated with high fat diets, and have been shown to lead tooxidative stress and cognitive impairment. (Morrison et al. (2010) J.Neurochem. 114:1581-1589).

GFAP (Glial fibrillary acidic protein) is a marker of neuronal damage.GFAP is an intermediate filament protein found almost exclusively inastrocytes which, in adults, control the level of GFAP expression.Astrocytes are a major type of glial cell which perform a variety ofstructural and metabolic functions, such as processingneurotransmitters, controlling extracellular ion levels, regulating thedirection and amount of nerve growth, maintaining the blood-brainbarrier, and participating in immune reactions. As astrocytes transformfrom a resting state into a process-bearing reactive state during eventssuch as aging, GFAP expression is up-regulated. GFAP levels have beenshown to increase in the brain tissue and cerebrospinal fluid inpatients suffering from Alzheimer's disease, and it has been suggestedthat reactive astrocytes may contribute to the neuropathology ofAlzheimer's disease (Wallin et al. (1996) Dementia 7:267). In theAlzheimer's diseased brain, the loss of synapses is associated with anincrease in the number of GFAP-positive astrocytes. In addition, thisloss of synapses appears to be related to the extent of reactiveastrogliosis (Brun et al. (1995) Neurodegeneration 4:171). GFAP is amajor component of the gliotic scars which result from gliosis, andwhich may interfere with subsequent reinnervation.

HNE (4-Hydroxynonenal) is a marker of oxidative stress, linked toAlzheimer's and Parkinson's disease. Increased levels of HNE have beendetected in brains with Alzheimer's disease (Markesbery et al. (1999)Brain Pathol 9(1):133-46; Sayre et al. (1997) J Neurochem68(5):2092-2097). HNE is an α,β-unsaturated aldehyde that is producedduring oxidation of membrane lipid polyunsaturated fatty acids. It isone of the major products of membrane peroxidation and is considered tobe largely responsible for cytotoxic effects observed under theoxidative stress. HNE exhibits variable adverse effects such asinhibition of DNA, RNA, and protein synthesis, interference with certainenzyme activities, and induction of heat shock proteins (Yoritaka et al.(1996) Proc. Natl. Acad. Sci. USA 93:2696-2701).

Parkinson's disease is a progressive disorder that affects a small areaof cells (called the substantia Nigra) in the middle part of the brainand which occurs slightly more often in men than women. It is alsocalled Shaking palsy or paralysis agitans and is a disorder of thecentral nervous system primarily attacking people between the ages 50and 69. Approximately one out of every 1,000 people contact the illness.One known cause of Parkinson's disease is the degeneration and death ofcells which normally produce dopamine, a chemical necessity fortransmitting messages in the brain. This causes a deficiency of dopamineand perhaps consequentially the symptoms of Parkinson's disease. Thecommon symptoms include tremor, stiffness (or rigidity) of muscles,slowness of movement (bradykinesia) and loss of balance (posturaldysfunction). Parkinson's disease is one of the most prevalentneurological conditions—along with epilepsy, stroke and dementia. Thenatural history of the disease results in a rate of progression from10-15 years from onset of the disease, to disability, with somevariability from patient to patient. Parkinson's itself, moreover, thedisability caused by the disease often leads to fatal infections such asaspiration, pneumonia, and urinary tract infections.

Parkinson's disease is usually categorized into three distinct groups.Paralysis agitans usually called Parkinson's disease is the most commonform of Parkinsonism, afflicting approximately 75% of the cases and isof unknown origin or cause. The second type of Parkinsonism which iscaused by drugs and toxins, which include carbon monoxide, manganese andchemical compound called MPTP (methyl-phenyltetrahydropyridine). Thethird form of Parkinsonism is called Vascular Parkinsonism which may becaused by multiple small strokes which damage the dopamine-producingbrain cells.

ADHD refers clinically to a relatively common syndrome (epidemiologicstudies have suggested that the prevalence of ADHD among the generalpopulation is between 2-10%). ADHD begins in childhood and typicallyremits by adulthood (Szatmari (1982) Child Adolesc. Psychial. Clin.North Am. 1:361-371). ADHD is clinically characterized by inattention(e.g. failure to give close attention, difficulties in sustainingattention, difficulties in organising tasks and activities and easilydistracted by extraneous stimuli), hyperactivity (e.g. difficulties inremaining seated, excessive motor activity in inappropriate situations,the patient acts as if “driven by a motor”), and impulsivity (e.g.difficulties in awaiting turn, answer questions before they have beencompleted and often interrupts or intrudes ongoing conversation).(American Psychiatric Association, Diagnostic and Statistical Manual ofMental Disorders (DSM-IV), 1994).

Chromium

Chromium is a nutritionally essential trace element. The essentiality ofchromium in the diet was established in 1959 by Schwartz. (Schwartz,“Present Knowledge in Nutrition,” page 571, fifth edition (1984, theNutrition Foundation, Washington, D.C.)). Chromium is essential foroptimal insulin activity in all known insulin-dependent systems (Boyleet al. (1977) Southern Med. J. 70:1449-1453). Chromium depletion ischaracterized by the disturbance of glucose, lipid and proteinmetabolism and by a shortened lifespan. Insufficient dietary chromiumhas been linked to both maturity-onset diabetes and to cardiovasculardisease.

Dietary supplementation of chromium to normal individuals has beenreported to lead to improvements in glucose tolerance, serum lipidconcentrations, including high-density lipoprotein cholesterol, insulinand insulin binding. (Anderson (1986) Clin. Psychol. Biochem. 4:31-41).Supplemental chromium in the trivalent form, e.g. chromic chloride, isassociated with improvements of risk factors associated with adult-onset(Type 2) diabetes and cardiovascular disease. Chromium supplementationhas been shown to reduce hyperglycemia, as well as promote weight loss,as described in U.S. Pat. Nos. 5,929,066, 6,329,361, and 6,809,115,which are each hereby incorporated by reference in their entirety. In aclinical study, Anderson et al. (Metabolism (1987) 36(4):351-355, 1987),chromium supplementation was shown to alleviate hypoglycemic symptomsand raise serum glucose levels out of the hypoglycemic range. In anotherstudy, chromium supplementation to overweight children with Type 1diabetes did not result in any cases of hypoglycemia (May, 2007). In yetanother study, chromium supplementation to adults with Type 1 diabetesdid not result in any cases of hypoglycemia; and allowed a 50% reductionin insulin dose (Ravina et al. (1995) J. Trace Elements in ExperimentalMed. 12:71-83).

The principal energy sources for the body are glucose and fatty acids.Chromium depletion results in biologically ineffective insulin andcompromised glucose metabolism. Under these conditions, the body reliesprimarily upon lipid metabolism to meet its energy requirements,resulting in the production of excessive amounts of acetyl-CoA andketone bodies. Some of the acetyl-CoA can be diverted to increasedcholesterol biosynthesis, resulting in hypercholesterolemia. Diabetesmellitus is characterized in large part by glycosuria,hypercholesterolemia, and often ketoacidosis. The acceleratedatherosclerotic process seen in diabetics is associated withhypercholesterolemia. (Boyle et al. (1977) Southern Med. J.70:1449-1453).

Chromium functions as a cofactor for insulin. It binds to the insulinreceptor and potentiates many, and perhaps all, of its functions. (Boyleet al. (1977) Southern Med. J. 70:1449-1453). These functions include,but are not limited to, the regulation of carbohydrate and lipidmetabolism. (Schwartz, “Present Knowledge in Nutrition,” page 571, fifthedition (1984, the Nutrition Foundation, Washington, D.C.)). Theintroduction of inorganic chromium compounds per se into individuals isnot particularly beneficial. Chromium must be converted endogenouslyinto an organic complex or must be consumed as a biologically activemolecule. Only about 0.5% of ingested inorganic chromium, however, isassimilated into the body. (Recommended Daily Allowances, Ninth RevisedEdition, The National Academy of Sciences, page 160, 1980). Only 1-2% ofmost organic chromium compounds are assimilated into the body.

U.S. Pat. Nos. 4,315,927 and Re. 33,988 disclose that when selectedessential metals, including chromium, are administered to mammals asexogenously synthesized coordination complexes of picolinic acid, theyare directly available for absorption without competition from othermetals. Describes therein are compositions and methods for selectivelysupplementing the essential metals in the human diet and forfacilitating absorption of these metals by intestinal cells. Thesecomplexes are safe, inexpensive, biocompatible, and easy to produce. Theexogenously synthesized essential metal coordination complexes ofpicolinic acid (pyridine-2-carboxylic acid) have the followingstructural formula:

wherein M represents the metallic cation and n is equal to the cation'svalence. For example, when M is Cr and n=3, then the compound is chromictripicolinate. Other chromium picolinates disclosed include chromicmonopicolinate and chromic dipicolinate.

The U.S. Recommended Daily Intake (RDI) of chromium is 120 μg. U.S. Pat.No. 5,087,623, the entire contents of which are hereby expresslyincorporated herein by reference, describes the administration ofchromic tripicolinate for the treatment of adult-onset diabetes in dosesranging from 50 to 500 pg. U.S. Pat. No. 6,329,361, the entire contentsof which are hereby expressly incorporated herein by reference,discloses the use of high doses of chromic tripicolinate (providing1,000-10,000 μg chromium/day) for reducing hyperglycemia and stabilizingthe level of serum glucose in humans with Type 2 diabetes. U.S. Pat.Nos. 5,789,401 and 5,929,066, the entire contents of which are herebyexpressly incorporated herein by reference, disclose a chromictripicolinate-biotin composition and its use in lowering blood glucoselevels in humans with Type 2 diabetes.

U.S. Pat. Nos. 5,087,623; 5,087,624; and 5,175,156, the entire contentsof which are hereby expressly incorporated herein by reference, disclosethe use of chromium tripicolinate for supplementing dietary chromium,reducing hyperglycemia and stabilizing serum glucose, increasing leanbody mass and reducing body fat, and controlling serum lipid levels,including the lowering of undesirably high serum LDL-cholesterol levelsand the raising of serum High Density Lipid (HDL)-cholesterol levels.U.S. Pat. Nos. 4,954,492 and 5,194,615, the entire contents of which arehereby expressly incorporated by reference, describe a related complex,chromic nicotinate, which is also used for supplementing dietarychromium and lowering serum lipid levels. Picolinic acid and nicotinicacid are position isomers having the following structures:

Nicotinic acid and picolinic acid form coordination complexes withmonovalent, divalent and trivalent metal ions and facilitate theabsorption of these metals by transporting them across intestinal cellsand into the bloodstream. Chromium absorption in rats following oraladministration of CrCl₃ was facilitated by the non-steroidalanti-inflammatory drugs (NSAIDs) aspirin and indomethacin. (Davis et al.(1995) J. Nutrition Res. 15:202-210; Kamath et al. (1997) J. Nutrition127:478-482). These drugs inhibit the enzyme cyclooxygenase whichconverts arachidonic acid to various prostaglandins, resulting ininhibition of intestinal mucus formation and lowering of intestinal pHwhich facilitates chromium absorption.

There remains a constant need for effective treatments of hypoglycemiaand hypoglycemia-related conditions. One such need is for safer and moreoptimal administrations of insulin. The present embodiments disclosedherein address this need by providing a safe, inexpensive, drug-freetherapeutic agent, and methods of administering the same.

SUMMARY

The embodiments disclosed herein are based, in part, upon the surprisingdiscovery of a novel chromium-insulin complex that has improvedtherapeutic efficacy and benefits. Thus, in accordance with theembodiments described herein, provided are compositions for the improveddelivery of insulin and/or chromium, and uses thereof.

Some embodiments relate to compositions comprising a chromium-insulincomplex. In some embodiments, the chromium-insulin complex comprise astoichiometric ratio of chromium to insulin, e.g., 2:1, 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. In some embodiments, the chromiumand insulin are present in the chromium insulin complexes innon-stoichiometric amounts, e.g., between 1 and 10 molecules of chromium(e.g., chromium complex), per insulin molecule, or per insulin hexamer.In some embodiments the complex has a molecular weight that is betweenabout 30 and 40 kDa, e.g., 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, or more.

The improved compositions comprising chromium-insulin complexes areuseful for the delivery of insulin to individuals in need thereof, e.g.,an individual that has a glucose metabolism disorder or condition, suchas diabetes or hypoglycemia. In some embodiments, the compositionscomprising a chromium-insulin complex exhibits improved absorption intothe bloodstream, as compared to uncomplexed insulin, or other insulincomplexes. In some embodiments, the compositions comprising achromium-insulin complex exhibits more rapid decrease in serum glucoselevels, when compared to uncomplexed insulin, or other insulincomplexes. In some embodiments, the compositions comprising achromium-insulin complex decreases weight loss associated with Type Idiabetes. In some embodiments, the compositions comprising achromium-insulin complex decreases weight gain associated with Type 2diabetes.

Accordingly, some embodiments disclosed herein relate to compositionscomprising chromium-insulin complexes. In some embodiments, the amountsof chromium and insulin in the composition are selected together toprovide a therapeutically effective amount of chromium and or insulin.In some embodiments, a synergistically effective amount of chromium andinsulin is provided to achieve greater than additive effect. In someembodiments, the chromium and insulin composition can be used to providea greater therapeutic effect to a patient in need thereof than insulinalone, or compared to other insulin complexes, such as zinc-insulin.

In some aspects, the synergistically effective amount of chromium in thecomposition can be between about 5 and 2,000 micrograms. In someaspects, the synergistically effective amount of insulin is betweenabout 1 unit and 500 units. In some aspects, the composition comprises aratio of chromium to insulin between about 0.001 micrograms of chromiumto units of insulin and 20 micrograms of chromium to units of insulin.In some aspects, the chromium is selected from the group of chromiumcomplexes consisting of chromium picolinate, chromic tripicolinate,chromium nicotinate, chromic polynicotinate, chromium chloride, chromiumhistidinate, chromium trihistidinate, and chromium yeasts. Preferably,the chromium comprises a chromium histidinate. In some aspects, thecomposition provides increased insulin receptor binding. The chromiumcan be dissolved in a solution of the insulin or the chromium can besuspended in a solution of the insulin.

In accordance with the embodiments disclosed herein, provided is animproved method of administering insulin to a subject in need thereof,comprising combining insulin and chromium to create a composition, andadministering the optimal dosage of the composition to the subject. Theamounts of insulin and chromium can be synergistically effectiveamounts. The composition can comprise a chromium-insulin complex. Insome embodiments, the subject has a glucose metabolism-related diseaseor disorder. In some aspects, the disease or disorder is selected fromthe group consisting of: diabetes, Alzheimer's disease, dementia, mildcognitive impairment (MCI), attention deficit hyperactive disorder(ADHD), Huntington's Disease, epilepsy, and Parkinson's Disease. In someembodiments, an optimal dosage of the composition comprising achromium-insulin complex is determined for administration to the subjectprior to administration of the composition to the subject.

In some embodiments, a method for making an injectable composition ofchromium and insulin is provided that comprises combining chromium andinsulin, thereby arriving at the injectable composition. The chromiumcan suspended in a solution of the chromium can be dissolved in asolution. The chromium and insulin injectable composition can comprise achromium insulin complex. The injectable composition can be a suspensionor a solution. In some aspects, the ratio of chromium to insulin isbetween 0.001 micrograms of chromium per unit of insulin and 100micrograms of chromium per unit of insulin. In some embodiments, thecomposition is administered intranasally.

In some embodiments, a method for stabilizing serum glucose levels in ansubject in need thereof is provided that comprises identifying a subjectwho is in need of insulin; and administering a composition comprisingchromium and insulin to the subject. In some embodiments, thecomposition of chromium and insulin comprises a chromium-insulincomplex. In some aspects, the composition of chromium and insulin isadministered parenterally. In other aspects, the composition of chromiumand insulin is administered orally. In some aspects, the composition ofchromium and insulin is administered pulmonarily. In some aspects, thecomposition of chromium and insulin is administered nasally. In someembodiments, the subject has diabetes. In some embodiments, the subjectis overweight. In some embodiments, the subject is identified as havingdiabetes-induced weight loss.

In some embodiments, use of a composition comprising chromium andinsulin for stabilizing serum glucose levels in a subject in needthereof is provided. In some embodiments, the composition of chromiumand insulin comprises a chromium-insulin complex. In some aspects, thecomposition of chromium and insulin is formulated for administration byinjection. In other aspects, the composition of chromium and insulin isformulated for oral or intranasal administration. In some embodiments,the subject has diabetes. In some embodiments, the subject isoverweight. In some embodiments, the subject is identified as havingdiabetes-induced weight loss.

Accordingly, in some embodiments, provided herein are methods to reducethe loss of weight associated with insulin administration, or stabilizethe weight, in diabetic individuals receiving insulin therapy. Alsoprovided are compositions comprising chromium and insulin for reducingloss of weight associated with insulin administration, stabilizingweight in diabetic individuals receiving insulin therapy. In someembodmeitns,t eh composition of chromium an insulin comprise anchromium-insulin complex. In some aspects the composition of chromiumand insulin is formulated for administration by injection.

In some embodiments, an improved method for stabilizing serum glucoselevels in a subject in need thereof is provided, wherein the improvementcomprises administering insulin to the subject in the form of acomposition comprising a chromium-insulin complex. In some embodiments,the composition comprises synergistically effective amounts of chromiumand insulin. The composition can be administered parenterally, orally,pulmonarily, or transdermally. In some aspects, the synergisticallyeffective amount of chromium is between about 300 and 1,000 micrograms.In some aspects, the synergistically effective amount of insulin isbetween about 5 units and 50 units. In some embodiments, the subject hasa glucose metabolism-related disease or disorder.

In some embodiments, an improved method of treating diabetes, e.g., Type1 or Type 2 diabetes, in a subject in need thereof with insulincomprises administering to the subject a composition comprising insulinand chromium. In some embodiments, the composition comprises achromium-insulin complex. In some embodiments, the compositioncomprising insulin and chromium is administered parenterally. In someembodiments, the composition is administered nasally. In someembodiments, the composition is administered pulmonary. In someembodiments, the composition is administered transdermally. Someembodiments provide a composition comprising chromium and insulin forthe treatment of diabetes, e.g., Type 1 or Type 2 diabetes. In someembodiments, the composition is formulated for administration byinjection. In some embodiments, the composition comprises achromium-insulin complex.

In some embodiments, method of preventing insulin-induced weight loss ina subject with diabetes comprises identifying a subject in need ofinsulin therapy for the treatment of diabetes administering to thesubject a composition comprising insulin and chromium. In some aspects,the composition comprising insulin and chromium is administeredparenterally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing serum glucose levels 0.5 hours after aninsulin injection, for control (no insulin) and treatment groups, asdescribed in Example 1. One treatment group was administered onlyinsulin (“Hypo” or “H”). Another treatment group was administeredinsulin and chromium picolinate (“H+CrPic”). The final group wasadministered insulin and chromium histidinate (“H+CrHis”).

FIG. 2 is a bar graph showing brain chromium levels after treatment forcontrol (no treatment) and treatment groups (H, H+CrPic, and H+CrHis),as described in Example 1.

FIG. 3 is a bar graph showing GLUT-1 transporter levels after treatmentfor control (no treatment) and treatment groups (H, H+CrPic, andH+CrHis), as described in Example 1.

FIG. 4 is a bar graph showing GLUT-3 transporter levels after treatmentfor control (no treatment) and treatment groups (H, H+CrPic, andH+CrHis), as described in Example 1.

FIG. 5 is a bar graph showing hippocampus Nrf2 (nuclear factor erythroid2 related factor 2) levels after treatment for control (no treatment)and treatment groups (H, H+CrPic, and H+CrHis), as described in Example1.

FIG. 6 is a bar graph showing hippocampus GFAP (glial fibrillary acidicprotein) & HNE (4-Hydroxynonenal) levels after treatment for control (notreatment) and treatment groups (H, H+CrPic, and H+CrHis), as describedin Example 1.

FIG. 7 is a bar and line graph showing the effect of insulin-chelatetype on glucose levels of type-1 diabetes induced rats, as described inExample 3. In addition to control (no treatment), five treatment groupswere respectively administered the following: streptozotocin (STZ);streptozotocin and zinc oxide (STR+Zn); streptozotocin and chromiumhistidinate (STZ+CrHis); streptozotocin, zinc oxide, and insulin(STR+ZnIns); and, streptozotocin, chromium histidinate, and insulin(STR+CrIns).

FIG. 8 is a bar graph showing kidney OCT-1 (organic cationtransporter 1) levels after treatment for control and treatment groups(STR, STR+Zn, STR+Cr, STR+ZnIns, and STR+CrIns), as described in Example3.

FIG. 9 is a bar graph showing kidney OCT-2 (organic cationtransporter 1) levels after treatment for control and treatment groups(STR, STR+Zn, STR+Cr, STR+ZnIns, and STR+CrIns), as described in Example3.

FIG. 10 is a bar graph showing kidney NFK (nuclear factor kappa B)levels after treatment for control and treatment groups (STR, STR+Zn,STR+Cr, STR+ZnIns, and STR+CrIns), as described in Example 3.

FIG. 11 is a bar graph showing kidney MRP2 (multidrug resistance protein2) levels after treatment for control and treatment groups (STR, STR+Zn,STR+Cr, STR+ZnIns, and STR+CrIns), as described in Example 3.

FIG. 12 is a bar graph showing brain NFK levels after treatment forcontrol and treatment groups (STR, STR+Zn, STR+Cr, STR+ZnIns, andSTR+CrIns), as described in Example 3.

FIG. 13 is a bar graph showing brain insulin levels after treatment forcontrol and treatment groups (STR, STR+Zn, STR+Cr, STR+ZnIns, andSTR+CrIns), as described in Example 3.

FIG. 14 is a graph showing UV absorbance (mAU) over time of chromiumhistidinate eluted through a sizing column.

FIG. 15 is a graph showing UV absorbance (mAU) over time of insulineluted through a sizing column.

FIG. 16 is a graph showing UV absorbance (mAU) over time of supernatantfrom a chromium insulin composition eluted through a sizing column.

FIG. 17 is a graph showing counts over time output from an inductivelycoupled plasma mass spectrometry (“ICPMS”) device targeting ⁵²Cr ofsupernatant from a chromium insulin composition eluted through a sizingcolumn.

FIG. 18 is a graph showing UV absorbance (mAU) over time of aredissolved precipitate from a chromium insulin composition elutedthrough a sizing column indicating the existence of a chromium-insulincomplex.

FIG. 19 is a graph showing counts over time from output from an ICPMSdevice targeting ⁵²Cr of a redissolved precipitate from a chromiuminsulin composition eluted through a sizing column indicating theexistence of a chromium-insulin complex.

FIG. 20 is a graph showing counts over time from output from an ICPMSdevice targeting ⁵³Cr of a redissolved precipitate from a chromiuminsulin composition eluted through a sizing column indicating theexistence of a chromium-insulin complex.

FIG. 21 is a line graph showing serum insulin levels over time for micetreated with either regular insulin (R-In), chromium insulin (Cr-In), orzinc-insulin (Znc-In).

FIG. 22 is a line graph showing serum glucose levels over time for micetreated with either regular insulin (R-In), chromium insulin (Cr-In), orzinc-insulin (Znc-In).

FIG. 23 is a line graph showing serum insulin levels over time fordiabetic mice treated with either saline (Control), regular insulin(Insulin), chromium insulin (Cr-In), or zinc-insulin (Znc-In).

FIG. 24 is a line graph showing serum glucose levels over time fordiabetic mice treated with either regular insulin (Insulin), chromiuminsulin (Cr-In), or zinc-insulin (Znc-In).

FIG. 25 is a bar graph showing serum glucose levels after treatment forcontrol and treatment groups (Control, Type 1, +ZnIns, +CrIns), asdescribed in Example 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The embodiments disclosed herein are based, in part, upon Applicant'sdiscovery of the unexpected protective effect of chromium in preventinghypoglycemia, and in preventing or ameliorating hypoglycemia associatedconditions, such as brain injury and the like, as well as Applicant'sdiscovery of improved methods of administering insulin therapy to thosein need thereof.

Chromium

As used herein, the term “chromium” refers to chromium chloride,chromium yeasts, as well as chromium complexes. Some chromium complexesuseful in the embodiments disclosed herein include, but are not limitedto, the following: chromium histidinate; chromium trihistidinate;chromium polyhistidinate; chromium dinicocysteinate; chromiumdinicotinate tryptophan; chromium dinicotinate tyrosine; chromiumdinicotinate hydroxycitrate; chromium dinicotinate cinnamate; chromiumdinicotinate gallate; chromium dinicotinate 5-hydroxytryptophan;chromium dinicotinate aspartate; chromium dinicotinate glutamate;chromium dinicotinate arginate; chromium tris(tryptophan); chromiumnicotinate, chromium polynicotinate; chromium picolinate; chromiummonopicolinate; chromium dipicolinate; chromium tripicolinate; chromiumtriphenylalanine; chromium tris(tyrosine); chromiumtris(hydroxycitrate); chromium tris(5-hydroxytryptophan); chromiumtris(cinnamate); chromium tris(gallate); chromium complexes disclosedherein are chromium having three different carboxylate ligands. Byvarying ligands from nicotinic acid, glutamate, cysteinate, aspartate,argininate, tyrosine and tryptophan, at least 30 possible chromiumcomplexes can be produced.

In various cases, the ligand(s) has/have the ability to bond to chromiumvia its carboxylate functional group as well as through pi electron-dorbital interaction. This secondary interaction between the ligand andchromium can increase the bioavailability and absorption of chromium.

In some embodiments, the chromium can be in the form of complexes oftrivalent chromium and at least one and no more than three tyrosine ortryptophan ligands. In specific embodiments, the chromium can be in theform of chromium complexes such as chromium (III) tris(tryptophan) andchromium (III) tris(tyrosine).

In some embodiments, the chromium complexes can be complexes oftrivalent chromium and one or more compounds extracted from plants.Non-limiting examples of plants from which these compounds can beextracted include plants such as genus Garcinia, Groffoniasimplicifolia, cinnamon bark, gallnuts, sumac, witch hazel, tea leaves,and oak bark. For example, in some embodiments, chromium can be providedin the form of chromium hydroxycitrate, chromium hydroxytryptophan,chromium cinnamate, and chromium gallate.

Preferably, the chromium is provided as a combination of chromiumpicolinate and chromium histidinate, or as a combination of chromiumnicotinate and chromium histidinate. In some preferred embodiments, thechromium is provided as chromium histidinate.

While the chromium complexes aid in the absorption of chromium byintestinal cells, in some embodiments, uncomplexed chelating agents areadvantageously included in the compositions to facilitate absorption ofother ingested chromium as well as other metals including, but notlimited to, copper, iron, magnesium, manganese, and zinc. Suitablechelating agents include histidine, any essential amino D or L aminoacids, tri amino acid formulae including but not limited to,triphenylalanine, tri histidine, tri arginine, picolinic acid, nicotinicacid, or both picolinic acid and nicotinic acid.

Chelating agents such as histidine, picolinic acid and nicotinic acidare available from many commercial sources, including Sigma-Aldrich (St.Louis, Mo.) (picolinic acid; catalog No. P5503; nicotinic acid; catalogNo. PN4126). In some embodiments, the ratio of the chromium complex tothe chelating agent in the embodiments disclosed herein can be fromabout 10:1 to about 1:10 (w/w), more preferably from about 5:1 to about1:5 (w/w), e.g., 5:1, 5:2, 5:3, 5:4, 1:1; 1:2, 1:3, 1:4, 1:5, or anynumber in between. Alternatively, the molar ratio of chromium complex tothe uncomplexed chelating agent is preferably 1:1, and can be from about5:1 to about 1:10, e.g., e.g., 5:1, 5:2, 5:3, 5:4, 1:1; 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any number in between. The chelatingagents with D or L amino acid and or with tri or mono and di forms ofchromium complex with tri amino acid or one or more amino acids but notlimited to chromium triphenylanine, chromium trihistidine, chromium polyphenylanine, chromium poly hisitidine, chromium polynicotinate, chromiumdi phenylananine, chromium di picolinic acid, chromium di hisitidineetc.

Some embodiments provide compositions and methods of treating subjectswith compositions that comprise or consist of a therapeuticallyeffective amount of chromium. Some embodiments provide compositions andmethods of treating subjects with compositions that comprise, consistessentially of, or consist of a therapeutically effective amount ofinsulin. Some embodiments provide compositions and methods of treatingsubjects with compositions that comprise, consist essentially of, orconsist of a therapeutically effective amount of chromium and atherapeutically effective amount of insulin. For example, someembodiments provide compositions and method of treating subjects thatcomprises, consists essentially of, or consist of a chromium-insulincomplex. Various methods of treatment are discussed below.

A “therapeutically effective amount” as used herein includes within itsmeaning a non-toxic but sufficient amount of a compound activeingredient or composition comprising the same for use in the embodimentsdisclosed herein to provide the desired therapeutic effect. The exactamount of the active ingredient disclosed herein required will vary fromsubject to subject depending on factors such as the species beingtreated, the age and general condition of the subject, the severity ofthe condition being treated, the particular agent being administered,the weight of the subject, and the mode of administration and so forth.Thus, it is not possible to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” may bedetermined by one of ordinary skill in the art using only routinemethods.

By way of example, a “therapeutically effective amount” of the chromiumdisclosed herein can be, for example, 0.001 μg/kg, 0.01 μg/kg, 0.1μg/kg, 0.5 μg/kg, 1 μg/kg, 1.5 μg/kg, 2.0 μg/kg, 2.5 μg/kg, 3.0 μg/kg,3.5 μg/kg, 4.0 μg/kg, 4.5 μg/kg, 5.0 μg/kg, 10 μg/kg, 15 μg/kg, 20μg/kg, 25 μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55μg/kg, 60 μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90μg/kg, 95 μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg, 250 μg/kg, 300 μg/kg,350 μg/kg, 400 μg/kg, 450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 650μg/kg, 700 μg/kg, 750 μg/kg, 80 μg/kg 0, 850 μg/kg, 900 μg/kg, 1 mg/kg,1.5mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3 mg/kg, 4.0mg/kg, 5.0 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg 50 mg/kg, 55 mg/kg, 60 mg/kg, 65mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100mg/kg, 125 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg,400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1 g/kg, 5g/kg, 10 g/kg, or more, or any fraction in between of chromium.Accordingly, in some embodiments, the dose of chromium in compositionsdisclosed herein can be about 0.001 μg to about 100 g, preferably perday. For example, the amount of chromium can be 0.001 μg 0.01 μg, 0.1μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg,25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg,225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg,450 μg, 475 μg, 500 μg, 525 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg,700 μg, 725 μg, 750 μg, 775 μg, 800 μg, 825 μg, 850 μg, 875 μg, 900 μg,925 μg, 950 μg, 975 μg, 1000 μg, 1.25 g, 1.5 g, 1.75 g, 2.0 g, 2.25 g,2.5 g, 2.75 g, 3.0 g, 3.25 g, 3.5 g, 3.5 g, 3.75 g, 4.0 g, 4.25 g, 4.5g, 4.75 g, 5.0 g, 5.25 g, 5.5 g, 5.75 g, 6.0 g, 6.25 g, 6.5 g, 6.75 g,7.0 g, 7.25 g, 7.5 g, 7.75 g, 8.0 g, 8.25 g, 8.5 g, 8.75 g, 9.0 g, 8.25g, 9.5 g, 9.75g, 10 g, 20g, 30g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100g, or more, or any range or amount in between any two of the precedingvalues. The exemplary therapeutically effective amounts listed above,can, in some embodiments be administered in the methods describedelsewhere herein on an hourly basis, e.g., every one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three hours, or any interval in between, or on adaily basis, every two days, every three days, every four days, everyfive days, every six days, every week, every eight days, every ninedays, every ten days, every two weeks, every month, or more or lessfrequently, as needed to achieve the desired therapeutic effect.

In some embodiments, a therapeutically effective amount of chromium isan amount that will reduce elevated blood glucose levels, but alsoprotects against hypoglycemia (e.g., reduced high glucose levels untilthey go down to normal, but the chromium does not enhance any furtherreduction below normal). In some embodiments, the compositions disclosedherein, e.g., compositions that comprise a chromium-insulin complex, canbe administered to a subject 1 time, 2 times, 3 times, 4 times 5 times,6 times, 7 times, 8 times, 9 times, 10 times, or more, per day, for aperiod of time, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months,5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months,1 year, or more, or any amount of time in between the preceding values.

In some embodiments, the compositions described herein, for examplecompositions that comprise chromium and insulin, e.g., achromium-insulin complex, can be administered to a subject per se, or inpharmaceutical compositions where they are mixed with other activeingredients, as in combination therapy, or suitable carriers orexcipient(s). Techniques for formulation and administration of thecompounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18thedition, 1990.

Insulin

As used herein, “insulin” refers to insulin from a variety of sources.Naturally occurring insulin and structurally similar bioactiveequivalents (insulin analogues including short acting and analogues withprotracted action) can be used. Insulin useful in the embodimentsdisclosed herein can be isolated from different species of mammal. Forexample, in some embodiments, animal insulin preparations extracted frombovine or porcine pancreas can be used. In some embodiments, insulinanalogues, derivatives and bioequivalents thereof can also be used. Inaddition to insulin isolated from natural sources, the embodimentsdisclosed herein can use insulin chemically synthesizing using proteinchemistry techniques such as peptide synthesis. In some embodiments,analogues of insulin are also suitable.

The insulin used in the embodiments disclosed herein may be obtained byisolating it from natural sources or by chemically synthesizing it usingpeptide synthesis, or by using the techniques of molecular biology toproduce recombinant insulin in bacteria or eukaryotic cells. Thephysical form of insulin may include crystalline and/or amorphous solidforms. In addition, dissolved insulin may be used. Synthetic forms ofinsulin are described in U.S. Pat. Nos. 4,421,685, 5,474,978, and5,534,488, the disclosure of each of which is hereby expresslyincorporated by reference in its entirety.

In some embodiments, the compositions provided herein comprise, consistessentially of, or consist of a combination of a therapeuticallyeffective amount of insulin and a therapeutically effective amount ofchromium. As discussed above, the exact amount of the chromium and/orinsulin will vary from subject to subject depending on factors such asthe species being treated, the age and general condition of the subject,the severity of the condition being treated, the particular agent beingadministered, the weight of the subject, and the mode of administration,and so forth. Thus, it is not possible to specify an exact“therapeutically effective amount”. However, for any given case, anappropriate “therapeutically effective amount” may be determined by oneof ordinary skill in the art using only routine methods. Exemplarydosage forms and therapeutically effective amounts of insulin useful inthe embodiments disclosed herein are described in, e.g. U.S. Pat. Nos.7,429,564 and 7,112,561, U.S. Patent Application Pub. No. 2010/0262434,and the like, each of which is hereby expressly incorporated byreference in its entirety.

By way of example, a “therapeutically effective amount” of the insulindisclosed herein can be, for example, 0.01 units of insulin, 0.1 unitsof insulin, 1 unit of insulin, 1.5 units of insulin, 2 units of insulin,3 units of insulin, 4 units of insulin, 5 units of insulin, 6 units ofinsulin, 7 units of insulin, 8 units of insulin, 9 units of insulin, 10units of insulin, 11 units of insulin, 12 units of insulin, 13 units ofinsulin, 14 units of insulin, 15 units of insulin, 16 units of insulin,17 units of insulin, 18 units of insulin, 19 units of insulin, 20 unitsof insulin, 21 units of insulin, 22 units of insulin, 23 units ofinsulin, 24 units of insulin, 25 units of insulin, 26 units of insulin,27 units of insulin, 28 units of insulin, 29 units of insulin, 30 unitsof insulin, 35 units of insulin, 40 units of insulin, 45 units ofinsulin, 50 units of insulin, 60 units of insulin, 70 units of insulin,80 units of insulin, 90 units of insulin, 100 units of insulin, 150units of insulin, 200 units of insulin, 250 units of insulin, 300 unitsof insulin, 400 units of insulin, 500 units of insulin, 1000 units ofinsulin, 2000 units of insulin, or more, or less, or any fraction inbetween.

Conventional administration of insulin is accomplished parenterally(e.g. intramuscularly, subcutaneously, intraperitoneal, etc.), however,there are numerous other methods of administration available that areuseful in the embodiments disclosed herein. U.S. Pat. No. 5,858,968, theentire contents of which are hereby expressly incorporated herein byreference, describes the administration of insulin orally, enterally(direct incubation into the stomach), or in an aerosol, i.e.,pulmonarily. U.S. Pat. No. 7,291,591, the entire contents of which arehereby expressly incorporated herein by reference, describes theadministration of insulin transdermally. U.S. Pat. No. 4,164,573, theentire contents of which are hereby expressly incorporated herein byreference, describes the administration of insulin rectally. U.S. Pat.No. 5,053,389, the entire contents of which are hereby expresslyincorporated herein by reference, describes various non-parenteral meansof administration of insulin, including ophthalmically (citing DanishPatent No. 135,268, the entire contents of which are hereby expresslyincorporated herein by reference). In some embodiments, the compositiondisclosed herein can be formulated for nasal administration, e.g., viaan atomizer or the like.

Parenteral Administration of Insulin

U.S. Patent Application Pub. No. 2010/0262434, the entire contents ofwhich are hereby expressly incorporated by reference, describes theprocess for determining the proper dose of injected insulin for a givenpatient. There are a number of factors that make the administration of aproper insulin dose difficult. First, injected insulin does not impactblood glucose instantly. Even fast acting insulin formulations takehours to have a biological effect. As such, conservative dosing canproduce hours of high glucose before supplemental injections can beapplied to reduce the glucose concentration. Over-dosing can result inhypoglycemia, which presents risk of acute incapacitation or coma.

Second, a varied diet requires a concomitant adjustment in insulindosage. The carbohydrates present in some foods is rapidly converted toglucose. The correct insulin dose, measured in units, U, necessary forthe body to utilize the glucose from the carbohydrate component of ameal, I_(C), is proportional to the carbohydrate intake, Carbs:

I _(C)=Carbs/CIR

Where CIR, the carbohydrate to insulin sensitivity factor, is particularfor each patient and may vary depending upon a patient's condition.

Third, when the blood glucose level, BG, is not near a patient's targetglucose level, BG_(T), before a meal begins or at a time after allinjected insulin has been utilized, adjustments (in the form of insulinor food depending on the direction of deviation) should be administeredto correct for the deviation. The amount of insulin adjustment for highblood glucose deviations, I_(B), depends on the patient's individualinsulin sensitivity factor, ISF.

I _(B)=(BG−BG_(r))/ISF

I_(B) can be positive if BG is higher than the target or negative if BGis lower than the target. If positive, a dosage of insulin I_(B) shouldreturn the patient near to their target blood glucose level. If I_(B) isnegative, the current blood glucose (BG) is below the target, so theadjustment would need to involve food ingestion.

Fourth, if I_(B) is negative, food can be consumed to effect anadjustment. Ideally, the amount of food would be just enough to correctthe low BG. A food intake sensitivity factor can be used to guide thefood intake. Basing the food intake on the food carbohydrate content iscurrently a preferred method. The recommended carbohydrate intake,Carbs, to correct for a given blood glucose negative deviation,BG-BG_(T) is:

Carbs=−CIR/ISF*(BG−BG_(T))

−CIR/ISF, is also known as 1/CGR, and can be calculated if one hasestimates for their CIR and ISF.

Fifth, the patient's sensitivity factors can be a function of theircondition. So, exercise, stress, illness, etc. can be sources ofvariation that change how the patient is utilizing insulin. Over longertime periods, the patient's weight and progressing conditions can impactthe sensitivity factors.

The ISF (insulin sensitivity factor) is the amount by which anindividual patient's blood glucose concentration is reduced for eachunit of rapid insulin taken. ISFs are generally in the range of 30 to 50mg/dL/U.

Oral Administration of Insulin

The oral administration of insulin is described in U.S. Pat. No.7,429,564, hereby expressly incorporated by reference in its entirety.Oral administration of insulin requires consideration of the samefactors affecting the parenteral administration of insulin, in additionto factors specific to oral administration, including, for example, thechemical structure of the particular delivery agent, the nature andextent of interaction of insulin and the delivery agent, the nature ofthe unit dose; the concentration of delivery agent in thegastrointestinal tract, and the ratio of delivery agent to insulin. Thenature of the unit dose for oral administration can be, but is notlimited to, solid, liquid, tablet, capsule, suspension, or otheracceptable dosage forms. The means of delivery of the pharmaceuticalcomposition (for example, a composition comprising chromium and insulin,e.g., a composition comprising a chromium-insulin complex), can be, butis not limited to, for example, a capsule, compressed tablet, pill,solution, gel, freeze-dried powder ready for reconstitution, suspensionsuitable for administration to the subject, or other means.

Typically, insulin is not absorbed through the gastrointestinal tract.However, there are several delivery agents that make insulinbioavailable and absorbable through the gastrointestinal mucosa whenorally administered. By way of example, an acceptable delivery agent caninclude, a compound of the formula or a pharmaceutically effective saltthereof:

wherein X is hydrogen or halogen; and R is substituted or unsubstitutedC₁-C₃ alkylene, substituted or unsubstituted C₁-C₃ alkenylene,substituted or unsubstituted C1-C3 alkyl (arylene), or substituted orunsubstituted C₁-C₃ aryl (alkylene). The acceptable delivery agents alsoinclude, but are not limited to, a compound of the formula above or apharmaceutically effective salt thereof wherein: X is a hydrogen orhalogen; and R is substituted or unsubstituted C₁-C₁₂ alkylene, orsubstituted or unsubstituted C₁-C₁₂ alkenylene. The acceptable deliveryagents also include, but are not limited to, a compound of the formulaabove or a pharmaceutically effective salt thereof wherein X is chlorineand R is C₃ alkylene. Acceptable insulin delivery agents can alsoinclude a compound of the formula or a pharmaceutically effective saltthereof:

wherein X is one or more of hydrogen, halogen, hydroxyl, or C₁-C₃alkoxy; and R is substituted or unsubstituted C₁-C₃ alkylene, orsubstituted or unsubstituted C₁-C₃ alkenylene. The acceptable deliveryagents also include the compound 4-[(4-chloro,2-hydroxybenzoyl)amino]butanoic acid (alternatively known asN-(4-chlorosalicyloyl)-4-aminobutyrate, or by the short name “4-CNAB”),as well as the monosodium salt thereof

In some embodiments, the delivery agents can be in the form of thecarboxylic acid or salts thereof. Suitable salts include, but are notlimited to, organic and inorganic salts, for example alkali-metal salts,such as sodium, potassium and lithium; alkaline-earth metal salts, suchas magnesium, calcium or barium; ammonium salts; basic amino acids, suchas lysine or arginine; and organic amines, such as dimethyl amine orpyridine. Preferably, the salts are sodium salts. The salts may be mono-or multi-valent salts, such as monosodium salts and di-sodium salts. Thesalts may also be solvates, including ethanol solvates, and hydrates.

Other suitable delivery agents that can be used for oral administrationof insulin include those delivery agents described U.S. Pat. Nos.5,650,386, 5,773,647, 5,776,888, 5,804,688, 5,866,536, 5,876,710,5,879,681, 5,939,381, 5,955,503, 5,965,121, 5,989,539, 5,990,166,6,001,347, 6,051,561, 6,060,513, 6,090,958, 6,100,298, 5,766,633,5,643,957, 5,863,944, 6,071,510 and 6,358,504, each of which is herebyexpressly incorporated by reference in its entirety. Additional suitabledelivery agents are described in International Publications Nos. WO01/34114, WO 01/21073, WO 01/41985, WO 01/32130, WO 01/32596, WO01/44199, WO 01/51454, WO 01/25704, WO 01/25679, WO 00/50386, WO02/02509, WO 00/47188, WO 00/07979, WO 00/06534, WO 98/25589, WO02/19969, WO 00/59863, WO 95/28838, WO 02/20466, WO 02/19969, WO02/069937, and WO 02/070438, each of which is hereby expresslyincorporated by reference in its entirety.

The insulin dose for oral administration is typically higher than forparenteral administration. The preferred amount of orally administeredinsulin varies from subject to subject, and can be determined by oneskilled in the art, taking into consideration factors such as thespecies being treated, the age and general condition of the subject,co-morbidities, the severity of the condition being treated, type ofinsulin being administered, the weight of the subject, the deliveryagent used, and so on.

Other Modes of Administration of Insulin

U.S. Pat. No. 7,112,561, hereby expressly incorporated by reference inits entirety, describes compositions and methods for delivery of insulinother than by injection, across skin, and membranes of various bodycavities such as ocular, nasal, oral, buccal, anal, rectal, vaginal,blood-brain barrier, and like membranes. Administration of insulinthrough skin membranes and membranes of body cavities requiresconsideration of the same factors affecting the parenteraladministration of insulin, in addition to other factors specific toadministration through skin membranes and/or membranes of body cavities,including, but not limited to: the chemical structure of the particulardelivery agent; the nature and extent of interaction of insulin and thedelivery agent; the nature of the unit dose; the concentration ofdelivery agent, and, the ratio of delivery agent to insulin. The natureof the unit dose for oral administration can be, but is not limited to,solid, liquid, tablet, capsule, or suspension. The means of delivery ofthe pharmaceutical composition can be, but is not limited to, forexample, a capsule, compressed tablet, pill, solution, freeze-dried,lotion, foam, aerosol, cream, gel, or powder ready for reconstitution orsuspension suitable for administration to the subject.

The delivery agent for administration through skin membranes ormembranes of body cavities can include permeation enhancers tofacilitate delivery of insulin through the membranes. The acceptablepermeation enhancers can include compounds having the followingstructure:

wherein X and Y are oxygen, sulfur or an imino group of the structure:

or ═N—R with the proviso that when Y is the imino group, X is an iminogroup, and when Y is sulfur, X is sulfur or an imino group, A is a grouphaving the structure:

wherein X and Y are defined above, m and n are integers having a valuefrom 1 to 20 and the sum of m+n is not greater than 25, p is an integerhaving a value of 0 or 1, q is an integer having a value of 0 or 1, r isan integer having a value of 0 or 1, and each of R, R₁, R₂, R₃, R₄, R₅,and R₆ is independently hydrogen or an alkyl group having from 1 to 6carbon atoms which may be straight chained or branched provided thatonly one of R, to R₆ can be an alkyl group, with the proviso that whenp, q and r have a value of 0 and Y is oxygen, m+n is at least 11, andwith the further proviso that when X is an imino group, q is equal to 1,Y is oxygen, and p and r are 0, then m+n is at least 11. Preferably, thepermeation enhancer defined above is combined in a composition with atherapeutically effective amount of insulin and a liquid carrier, saidcomposition having an acidic pH. In general, the pH of the compositionis at least 2 and no greater than 4.5. Preferably, the pH is: no greaterthan 4. More preferably, the pH is in the rage of 2.5 to 3.8. Even morepreferably, the pH is about 3.

Other suitable permeation enhancers are described in U.S. Pat. Nos.5,023,252, and 5,731,303 which are hereby expressly incorporated byreference in its entirety.

Although the above are preferred permeation enhancers, one of ordinaryskill in the art would recognize that the instant teachings would alsobe applicable to other permeation enhancers. Non-limiting examples ofother permeation enhancers useful in the embodiments disclosed hereinare the simple long chain esters that are Generally Recognized As Safe(GRAS) in the various pharmacopoeial compendia. These may include simplealiphatic, unsaturated or saturated (but preferably fully saturated)esters, which contain up to medium length chains. Non-limiting examplesof such esters include isopropyl myristate, isopropyl palmitate,myristyl myristate, octyl palmitate, and the like. The enhancers are ofa type that are suitable for use in a pharmaceutical composition. Theartisan of ordinary skill will also appreciate that those materials thatare incompatible with or irritating to mucous membranes should beavoided.

The enhancer can be present in the composition in a concentrationeffective to enhance penetration of the insulin, to be delivered,through the membrane. Various considerations should be taken intoaccount in determining the amount of enhancer to use. Suchconsiderations include, for example, the amount of flux (rate of passagethrough the membrane) achieved and the stability and compatibility ofthe components in the formulations. The enhancer is generally used in anamount of about 0.01 to about 25 wt. % the composition, more generallyin an amount of about 0.1 to about 15 wt. % the composition, and inpreferred embodiments in an amount of about 0.5 to about 15 wt % thecomposition.

The liquid carrier is present in the composition in a concentrationeffective to serve as a suitable vehicle for the compositions of theembodiments disclosed herein. In general, the carrier can be used in anamount of about 40 to about 98 wt. % of the composition and in preferredembodiments in an amount of about 50 to about 98 wt. % of thecomposition.

In general, compositions that contain insulin can be stored in arefrigerator. However, refrigeration may result in crystallization ofthe permeation enhancer. In order to inhibit or prevent suchcrystallization, in a preferred embodiment the composition includes oneor more crystallization inhibitors to inhibit the crystallization of thepermeation enhancer. Crystallization, if allowed to proceed, renders theemulsion unstable and has an adverse effect on shelf life. Preferredcrystallization inhibitors function by lowering the temperature at whichthe involved compound crystallizes. Examples of such crystallizationinhibitors include natural oils, oily substances, waxes, esters, andhydrocarbons. Examples of natural oils or oily substances includeVitamin E acetate, octyl palmitate, sesame oil, soybean oil, saffloweroil, avocado oil, palm oil, and cottonseed oil. The selection of asuitable crystallization inhibitor is deemed to be within the scope ofthose skilled in the art from the teachings herein. Preferredcrystallization inhibitors function by lowering the temperature at whichthe permeation enhancer crystallizes.

Inhibitors which are capable of lowering the temperature ofcrystallization of the involved compound to below about 25° C., areparticularly preferred, with those capable of lowering thecrystallization of the involved compound to below about 5° C. beingespecially preferred. Examples of especially preferred crystallizationinhibitors for use in inhibiting the crystallization ofoxacyclohexadecan-2-one include hexadecane, isopropyl myristate, octylpalmitate, cottonseed oil, safflower oil, and Vitamin E acetate, each ofwhich may be used in pharmaceutical preparations.

The crystallization inhibitor is present in the composition in aconcentration effective to inhibit the crystallization of the permeationenhancer. In general the crystallization inhibitor is present in anamount of about 0.001 to about 5 wt. % the composition, more generallyin an amount of from about 0.01 to about 2 wt % the composition. In oneembodiment the crystallization inhibitor is present in an amount of fromabout 0.1 to about 1 wt. % of the composition. The crystallizationinhibitor is one preferably used when the enhancer has a crystallizationtemperature above about 0° C. In particular, for example, acrystallization inhibitor is preferably used when the enhancer is,pentadecalactone and/or cyclohexadecanone, since these crystallize aboveroom temperature.

Compositions Comprising Chromium and Insulin

Zinc ions have been reported exert a stabilizing effect on insulinsolutions. See, e.g., U.S. Pat. No. 4,476,118, incorporated by referenceit its entirety. For example, including two to five zinc ions perhexamer of insulin may help prevent insulin precipitation. In constrastto the Zn-insulin complexes described, in U.S. Pat. No. 4,476,118, thechromium-insulin complexes provide favorable absorption and therapeuticeffects.

In some embodiments, chromium is provided in combination with insulin,e.g., within a single dosage form, such as a single injectable dosageform or a single oral dosage form. In some embodiments, chromium isprovided with insulin in a multi-unit dosage form. In some embodiments,chromium is provided dissolved in an insulin solution. In otherembodiments, chromium is provided in suspension in an insulin solution.Accordingly, provided herein are compositions that comprise, consistessentially of, or consist of chromium and insulin. In some embodiments,the compositions comprise a chromium-insulin complex.

In some embodiments, the compositions provided herein include acombination of insulin and chromium, e.g., within in a single dosageform, and are formulated for intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectal, ophthalmic, or topical delivery. In some embodiments, chromiumis provided with insulin in a multi-unit dosage form. In someembodiments, chromium is provided in suspension in an insulin solution.Intranasal delivery may be accomplished with an atomizer. The preferredmode of administration is left to the discretion of the practitioner,and will depend in part upon the site of the medical condition. Theeffective amounts of chromium, insulin, and or chromium-insulin complexcan vary depending on the route of administration. In most instances,administration will result in the release of the compounds of theembodiments disclosed herein into the bloodstream. Accordingly, providedherein are compositions that comprise, consist essentially of, orconsist of chromium and insulin, for example, in the form of achromium-insulin complex.

In some embodiments, the compositions provided herein comprise, consistessentially of, or consist of a combination of a therapeuticallyeffective amount of insulin and a therapeutically effective amount ofchromium. In some embodiments, these compositions can comprise, consistessentially of, or consist of a chromium-insulin complex. In someembodiments, the compositions provided herein comprise, consistessentially of, or consist of a combination of a synergisticallyeffective amount of insulin and a synergistically effective amount ofchromium. In some embodiments, the se compositions can comprise, consistessentially of, or consist of a chromium insulin complex. In someembodiments, the compositions provided herein comprise chromium that isdissolved in a solution of insulin. In some embodiments, the secompositions can comprise, consist essentially of, or consist of achromium insulin complex. In other embodiments, the compositionsprovided herein comprise chromium that is suspended in a solution ofinsulin. In some embodiments, the se compositions can comprise, consistessentially of, or consist of a chromium insulin complex.

In some embodiments, the compositions provided herein include aninjectable solution comprising a combination of a therapeuticallyeffective amount of chromium and a therapeutically effective amount ofinsulin. In some embodiments, the combination of chromium and insulinresults in chemical structures that have benefits for the treatment ofdiabetes, including but not limited to increased rate of absorption andoverall absorption of insulin,decreased insulin dissolution rate,increased receptor binding, and therapeutic properties. In someembodiments, the combination of chromium and insulin reduces serumglucose levels at a faster rate than insulin alone.]

Chromium-Insulin Complexes

In some embodiments, the chromium and insulin compositions providedherein comprise, consist essentially of, or consist of achromium-insulin complex. In some embodiments, the chromium-insulincomplex comprise a stoichiometric ratio of chromium to insulin, e.g.,1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. In someembodiments, the chromium and insulin are present in the chromiuminsulin complexes in non-stoichiometric amounts, e.g., between 1 and 10molecules of chromium (e.g., chromium complex), per insulin hexamer. Insome embodiments the complex has a molecular weight that is betweenabout 30 and 40 kDa, e.g., 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, or more.

In some embodiments, the chromium-insulin complex provides atherapeutically effective amount of chromium and insulin. In someembodiments, the composition comprises, consists essentially of, orconsists of isolated and/or purified amounts of a chromium-insulincomplex. The isolated and/or purified amounts of a chromium-insulincomplex can be provided in amounts to provide a therapeutically effectamount of chromium and/or insulin. In some embodiments, thechromium-insulin complex is not isolated and/or purified, but rather ispresent within a mixture of chromium and insulin. [0105] In someembodiments, chromium and/or insulin are provided with a nutritionallyacceptable carrier or a pharmaceutically acceptable carrier. As usedherein, the phrase “nutritionally acceptable carrier”, “nutritionallyacceptable excipient”, “pharmaceutically acceptable carrier”, or“pharmaceutically acceptable excipient” refers to nutritionally orpharmaceutically acceptable materials, compositions or vehicles,suitable for administering compounds of the embodiments disclosed hereinto mammals. The carriers can include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Carriers can be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as nutritionally or pharmaceutically acceptable carriersinclude, but are not limited to: sugars, such as lactose, glucose andsucrose; starches, such as corn starch and potato starch; cellulose, andits derivatives, such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients; such as cocoa butter and suppository waxes; oils, such aspeanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cornoil and soybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. In some embodiments, the nutritionally orpharmaceutically acceptable carrier can be suitable for intravenousadministration. In some embodiments, the nutritionally orpharmaceutically acceptable carrier can be suitable for locoregionalinjection.

The language “pharmaceutical composition” is used interchangeably with“therapeutic agent” and includes preparations suitable foradministration to mammals, e.g., humans. When the compounds of theembodiments disclosed herein are administered as pharmaceuticals tomammals, e.g., humans, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1 to 99.5% (more preferably, 0.5to 90%) of active ingredients in combination with a nutritionally orpharmaceutically acceptable carrier. The amount of therapeutic agentsincorporated into the multiple unit dosage form of the embodimentsdisclosed herein is quantum sufficiat to achieve the desired therapeuticeffect. The dosage amounts for the disclosed therapeutic agents arewell-established in the arts and can be optimized for any particularindication via routine experimentation.

In another aspect, the embodiments relate to methods of treating asubject with the compositions disclosed herein. The terms “subject,”“patient” or “individual” as used herein refer to a vertebrate,preferably a mammal, more preferably a human. “Mammal” can refer to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sport, or pet animals, such as, for example, horses,sheep, cows, pigs, dogs, cats, etc. Preferably, the mammal is human.

Synergistically Effective Compositions Comprising Chromium and Insulin

In some embodiments, the compositions provided herein comprise asynergistically effective amount of chromium and insulin selectedtogether to provide a greater than additive effect. This greater thanadditive effect can include, but is not limited to, an increase ininsulin receptor binding. A “synergistically effective amount” as usedherein refers to the amount of one component of a composition necessaryto elicit a synergistic effect in another component present in thecomposition. A “synergistic effect” as used herein refers to a resultthat is markedly greater than what would be expected when eithercomponent is administered alone. The exact synergistically effectiveamounts of the active ingredients disclosed herein required will varyfrom subject to subject depending on factors such as the species beingtreated, the age and general condition of the subject, co-morbidities,the severity of the condition being treated, the particular agents beingadministered, the weight of the subject, and the mode of administration,and so forth. Thus, it is not possible to specify an exact “synergisticamount”. However, for any given case, an appropriate “synergisticallyeffective amount” may be determined by one of ordinary skill in the artusing routine methods.

By way of example, a “synergistically effective amount” of the chromium,e.g., present in the form of a chromium complex, disclosed herein canbe, for example 0.001 μg/kg, 0.01 μg/kg, 0.1 μg/kg, 0.5 μg/kg, 1 μg/kg,1.5 μg/kg, 2.0 μg/kg, 2.5 μg/kg, 3.0 μg/kg, 3.5 μg/kg, 4.0 μg/kg, 4.5μg/kg, 5.0 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25 μg/kg, 30 μg/kg, 35μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55 μg/kg, 60 μg/kg, 65 μg/kg, 70μg/kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95 μg/kg, 100 μg/kg, 150μg/kg, 200 μg/kg, 250 μg/kg, 300 μg/kg, 350 μg/kg, 400 μg/kg, 450 μg/kg,500 μg/kg, 550 μg/kg, 600 μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 80μg/kg 0, 850 μg/kg, 900 μg/kg, 1 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg,3 mg/kg, 4.0mg/kg, 5.0 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45mg/kg 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg,850 mg/kg, 900 mg/kg, 950 mg/kg, 1 g/kg, 5 g/kg, 10 g/kg, or more, orany fraction in between of chromium. Accordingly, in some embodiments,the synergistically effective amount of chromium in compositionsdisclosed herein can be about 0.001 μg to about 1 g, preferably per day.For example, the amount of chromium, e.g., present in a chromiumcomplex, can be 0.001 0.01 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg,0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg,50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg,100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg,325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg,575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 725 μg, 750 μg, 775 μg,800 μg, 825 μg, 850 μg, 875 μg, 900 μg, 925 μg, 950 μg, 975 μg, 1000 μg,1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25mg, 3.5 mg, 3.5 mg, 3.75 mg, 4.0 mg, 4.25 mg, 4.5 mg, 4.75 mg, 5.0 mg,5.25 mg, 5.5 mg, 5.75 mg, 6.0 mg, 6.25 mg, 6.5 mg, 6.75 mg, 7.0 mg, 7.25mg, 7.5 mg, 7.75 mg, 8.0 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9.0 mg, 8.25 mg,9.5 mg, 9.75mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg,90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 1 g, or more, or anyrange or amount in between any two of the preceding values.

Likewise, by way of example, a “synergistically effective amount” ofinsulin disclosed herein can be, for example, 0.01 units of insulin, 0.1units of insulin, 1 unit of insulin, 1.5 units of insulin, 2 units ofinsulin, 3 units of insulin, 4 units of insulin, 5 units of insulin, 6units of insulin, 7 units of insulin, 8 units of insulin, 9 units ofinsulin, 10 units of insulin, 11 units of insulin, 12 units of insulin,13 units of insulin, 14 units of insulin, 15 units of insulin, 16 unitsof insulin, 17 units of insulin, 18 units of insulin, 19 units ofinsulin, 20 units of insulin, 21 units of insulin, 22 units of insulin,23 units of insulin, 24 units of insulin, 25 units of insulin, 26 unitsof insulin, 27 units of insulin, 28 units of insulin, 29 units ofinsulin, 30 units of insulin, 35 units of insulin, 40 units of insulin,45 units of insulin, 50 units of insulin, 60 units of insulin, 70 unitsof insulin, 80 units of insulin, 90 units of insulin, 100 units ofinsulin, 150 units of insulin, 200 units of insulin, 250 units ofinsulin, 300 units of insulin, 400 units of insulin, 500 units ofinsulin, 1000 units of insulin, 2000 units of insulin, or more, or anyfraction in between.

In other embodiments, there is a range of ratios of chromium to insulinthat results in the greatest synergistic effect upon the subject. Theexact ratio of the active ingredients disclosed herein required willvary from subject to subject depending on factors such as the speciesbeing treated, the age and general condition of the subject,co-morbidities, the severity of the condition being treated, theparticular agents being administered, the weight of the subject, and themode of administration, and so forth. Thus, it is not possible tospecify an exact ratio or range of ratios. However, for any given case,an appropriate ratio or range of ratios may be determined by one ofordinary skill in the art using only routine methods.

By way of example, the ratio of chromium to insulin disclosed herein canbe, for example, 0.0001 μg, chromium/unit insulin, 0.001 μg,chromium/unit insulin, 0.002 μg, chromium/unit insulin, 0.003 μgchromium/unit insulin, 0.004 μg chromium/unit insulin, 0.005 μgchromium/unit insulin, 0.006 μg chromium/unit insulin, 0.007 μgchromium/unit insulin, 0.008 μg chromium/unit insulin, 0.009 μgchromium/unit insulin, 0.01 μg chromium/unit insulin, 0.02 μgchromium/unit insulin, 0.03 μg chromium/unit insulin, 0.04 μgchromium/unit insulin, 0.05 μg chromium/unit insulin, 0.06 μgchromium/unit insulin, 0.07 μg chromium/unit insulin, 0.08 μgchromium/unit insulin, 0.09 μg chromium/unit insulin, 0.10 μgchromium/unit insulin, 0.11 μg chromium/unit insulin, 0.12 μgchromium/unit insulin, 0.13 μg chromium/unit insulin, 0.14 μgchromium/unit insulin, 0.15 μg chromium/unit insulin, 0.16 μgchromium/unit insulin, 0.17 μg chromium/unit insulin, 0.18 μgchromium/unit insulin, 0.19 μg chromium/unit insulin, 0.2 μgchromium/unit insulin, 0.3 μg chromium/unit insulin, 0.4 μgchromium/unit insulin, 0.5 μg chromium/unit insulin, 0.6 μgchromium/unit insulin, 0.7 μg chromium/unit insulin, 0.8 μgchromium/unit insulin, 0.9 μg chromium/unit insulin, 1 μg chromium/unitinsulin, 2 μg chromium/unit insulin, 3 μg chromium/unit insulin, 4 μgchromium/unit insulin, 5 μg chromium/unit insulin, 10 μg chromium/unitinsulin, 20 μg chromium/unit insulin, 50 μg chromium/unit insulin, 100μg chromium/unit insulin, 200 μg chromium/unit insulin, 500 μgchromium/unit insulin , or more, or any fraction in between. By way ofexample, the range of ratios of chromium to insulin disclosed herein canbe, for example, 0.001-20 μg chromium/unit insulin, 0.001-0.01 μgchromium/unit insulin 0.01-0.1 μg chromium/unit insulin, 0.1-1 μgchromium/unit insulin, 1-2 μg chromium/unit insulin, 2-3 μgchromium/unit insulin, 3-4 μg chromium/unit insulin, 4-5 μgchromium/unit insulin, 5-10 μg chromium/unit insulin, 10-20 μgchromium/unit insulin, or more, or any fraction in between.

In some embodiments, an improved method of administering insulin to asubject in need thereof is provided. This improved method comprises:combining synergistically effective amounts of insulin and chromium tocreate a composition; determining an optimal dosage of the compositionfor the subject; and, administering the optimal dosage of thecomposition to the subject. The optimal dosage of the composition can bedetermined by administering the composition to a subject, and thenadjusting the insulin dosage to the lowest value that achieves thedesired effect. For example, in treating diabetes, the desired effect isthe stabilization of serum glucose to a level that is neitherhyperglycemic nor hypoglycemic. Thus, the optimal dosage of acomposition for treating diabetes is the lowest dosage that brings serumglucose to a level that is neither hyperglycemic nor hypoglycemic. Givenchromium's serum glucose-stabilizing effect, the optimal dosage of acomposition comprising synergistically effective amounts of insulin andchromium likely contains less insulin than would be optimal were onlyinsulin being administered.

Kits Comprising Insulin and Chromium

In another aspect, the embodiments relate to an insulin injection kitcomprising a syringe, a solution of insulin, and chromium. In someembodiments, the kit includes a chromium-insulin complex. The kit allowsthe end user to combine the chromium and insulin prior toadministration. Thus, the end user is able to vary the dosage ofchromium and insulin, as well as the ratio of chromium to insulin in acomposition prior to injection. In some embodiments, the syringeincluded in the kit is configured to combine the insulin and chromiumwithin the syringe itself, such as the syringe disclosed in U.S. Pat.No. 4,424,057, the disclosure of which is hereby expressly incorporatedby reference in its entirety.

Methods for Making a Composition Comprising Chromium and Insulin

In another embodiment, the method for making an injectable compositionof chromium and insulin disclosed herein comprises combining chromiumand insulin, thereby arriving at the injectable composition. In someembodiments, the chromium component takes the form of, but is notlimited to: chromium (e.g., a chromium complex) suspended in a solution;chromium(e.g., a chromium complex) dissolved in a solution; chromium(e.g., in the form of a chromium complex) in a powder; or,chromium(e.g., a chromium complex) in any form combinable with asolution of insulin. In some embodiments, the injectable compositiontakes the form of, but is not limited to: a suspension; a solution; or,any other composition of chromium and a solution of insulin.

Methods for Treating Overweight Subjects in Need of Insulin

In another embodiment, a method for stabilizing serum glucose levels inan overweight subject in need thereof is provided, comprising the stepsof: (a) identifying an overweight subject who is in need of insulin,and, (b) administering a composition comprising chromium and insulin(e.g., in the form of a composition comprising a chromium-insulincomplex) to the subject. In some embodiments, the composition may beadministered parenterally, nasally, orally, or pulmonarily. In someembodiments, the overweight subject has diabetes, often Type 2 diabetes.In some embodiments, the subject has Type 1 diabetes. In other words, insome embodiments, the compositions and/or complexes described herein canbe used to stabilize serum glucose levels in subjects in need thereof.

Excess body weight is one of the major risk factor for developingdiabetes. Overweight people with diabetes overwhelmingly suffer fromType 2 diabetes. However, treatment of diabetes, e.g., with insulin,often results in increased body mass as a result of lowered metabolicrates and increased fat and glucose storage. This can lead to a cycle inwhich one's diabetes may worsen as a result of the weight gain caused bytreatment, leading to the need for more treatment, resulting in moreweight gain. By combining chromium and insulin, the weight gainassociated with insulin treatment can be attenuated by the weight losseffects of chromium supplementation. Chromium stabilizes serum glucoselevels above hypoglycemic levels, whereas insulin alone may causeexcessive glucose uptake, sometimes resulting in hypoglycemia. Becausechromium combined with insulin may result in less glucose uptakecompared to insulin alone, less glucose can be stored, resulting in lessweight gain. Likewise, because chromium works to normalize glucosemetabolism, administration of chromium with insulin may act to increasea subject's body weight where the subject is underweight.

In other embodiments, a method of preventing diabetes treatment-inducedweight gain is provided, comprising administering a compositioncomprising insulin and chromium. In some embodiments, the compositioncomprising insulin and chromium is administered parenterally.

Improved Method for Stabilizing Serum Glucose Levels

In another embodiment, an improved method for stabilizing serum glucoselevels in a subject in need thereof is provided. The improvementcomprises the steps of combining synergistically effective amounts ofinsulin and chromium to create a composition, and administering thecomposition to the subject. In some embodiments, the improved method isadministered, for example, parenterally, nasally, orally, pulmonarily,or transdermally. In some embodiments, the improvement comprisesproviding a therapeutically effective amount of a composition comprisinga chromium-insulin complex and administering the composition to thesubject. In this way, the compositions described herein can be used toprovide an improved method for stabilizing serum glucose levels incomparison to other known compositions.

Improved Method for Raising Serum Insulin Levels

In another embodiment, an improved method for raising serum insulinlevels is provided. The improvement comprises the steps of combiningsynergistically effective amounts of insulin and chromium to create acomposition, and administering the composition to the subject. In someembodiments, the improved method is administered, for example,parenterally, nasally, orally, pulmonarily, or transdermally. In someembodiments, the improvement comprises providing a therapeuticallyeffective amount of a composition comprising a chromium-insulin complexand administering the complex to the subject. That is to say, thecompositions described herein can be used to provide an improved methodfor elevating serum insulin levels in comparison to other knowncompositions and/or insulin alone.

Improved Method for Stabilizing Body Weight

In another embodiment, an improved method for stabilizing a subject'sbody weight is provided. In individuals with Type 1 diabetes, insulintherapy can result in weight loss. As shown in Example 6, below, thecompositions provided herein have been shown to reduce the weight lossassociated with insulin therapy in subjects with Type 1 diabetes.Individuals with Type 2 diabetes often experience weight gain. Thecompositions provided herein advantageously reduce weight gainassociated with Type 2 diabetes. In other words, the compositionsprovided herein are beneficial in stabilizing weight in individuals withdiabetes. The improvement comprises the steps of combiningsynergistically effective amounts of insulin and chromium to create acomposition, and administering the composition to a subject in needthereof. In some embodiments, the improved method is administered, forexample, parenterally, nasally, orally, pulmonarily, or transdermally.In some embodiments, the improvement comprises providing atherapeutically effective amount of a composition comprising achromium-insulin complex and administering the complex to the subject.In this way, the compositions described herein can be used to provide animproved method for elevating serum insulin levels in comparison toother known compositions and/or insulin alone.

Compositions and Methods for Treatment of OtherGlucose-Metabolism-related Diseases and Disorders

In another aspect, some embodiments relate to compositions for thetreatment of glucose metabolism-related diseases and disorders otherthan hypoglycemia, and methods of using the same. The compositions forthe treatment of other glucose metabolism related diseases are the samecompositions described herein, including chromium, and chromium incombination with insulin. Glucose metabolism-related diseases anddisorders include, but are not limited to, Alzheimer's Disease,dementia, mild cognitive impairment, attention deficit hyperactivitydisorder (ADHD), Parkinson's Disease, Huntington's Disease, AmyotrophicLateral Sclerosis (ALS), epilepsy, diabetes, hypoglycemia, and any otherglucose metabolism-related diseases and disorders. Thus, thecompositions and/or complexes disclosed herein may be used to treatglucose metabolism-related diseases such as Alzheimer's Disease,dementia, mild cognitive impairment, attention deficit hyperactivitydisorder (ADHD), Parkinson's Disease, Huntington's Disease, AmyotrophicLateral Sclerosis (ALS), epilepsy, diabetes, hypoglycemia.

EXAMPLES Example 1 Chromium Reduces the Severity of Brain Damage inInsulin-induced Hypoglycemic Rats

In order to evaluate chromium's potential protective effects preventinginsulin-induced hypoglycemia, animals were administered insulin toinduce hypoglycemia, and markers of hypoglycemic brain damage werecompared in animals with and without administration of chromium.

Briefly, hypoglycemia was induced in Sprague-Dawley rats (males, 8-weeksold) by intraperitoneal injection of 15U insulin/kg BW. The rats wereseparated into four groups of 15 rats each: (1) a control group notreceiving insulin (“Control”); (2) a group not administered chromium(“Hypo”); (3) a group administered 110 μg/kg/day of chromium picolinate(CrPic); and, a group administered 110 μg/kg/day of chromium histidinate(CrHis). After one week of dosing, brains were removed from thesacrificed rats and analyzed for markers of hypoglycemic damage: GLUT-1;GLUT-3; Nrf2; GFAP; and HNE. The data are shown in Table 1 and FIGS.1-6.

As shown in FIG. 1, chromium pretreatment did not raise or lower serumglucose levels after insulin injection. These data demonstrate thatchromium is useful for the normalization of serum glucose levels, i.e.,as influenced by insulin administration.

As shown in FIG. 2, in non-chromium treated animals, insulin inducedhypoglycemia significantly lowered brain chromium tissue levels. Bycontrast, chromium treatment raised brain chromium levels.

As shown in FIG. 3, hypoglycemia significantly raised brain GLUT-1transporter levels. The hypoglycemia-induced GLUT-1 increase was reducedin animals that received chromium treatment. These data suggest thatchromium may play a protective role by regulating GLUT-1 levels in orderthat excessive glucose does not enter the cells, which may lead to celldamage.

As shown in FIG. 4, hypoglycemia significantly raised brain GLUT-3transporter levels. The hypoglycemia-induced GLUT-3 increase was reducedin animals that received chromium treatment. These data suggest thatchromium may play a protective role by regulating GLUT-3 levels in orderthat excessive glucose does not enter the cells, which may lead to celldamage.

As shown in FIG. 5, hypoglycemia significantly lowered brain Nrf2 tissuelevels (cytoprotective protein). The hypoglycemia-induced Nrf2 decreasewas reduced by chromium treatment. As reduced Nrf2 levels have beenimplicated in cognitive impairment, these data demonstrate theusefulness of chromium in protecting against hypoglycemia-relatedconditions and disorders, including cognitive dysfunction.

As shown in FIG. 6, hypoglycemia significantly raised brain GFAP and HNEtissue levels (markers of neuronal and oxidative damage). GFAP and HNElevels were lowered by chromium treatment. These data demonstrate theusefulness of chromium in protecting against hypoglycemia-relatedconditions, including neuronal damage.

TABLE 1 Comparison of Hypoglycemic Brain Damage Markers (mean ± s.d.)Control Hypo CrPic CrHis Serum Glucose at 0.5 hrs 115.6 ± 5.5    42.6 ±6.5  40.7 ± 5.2  42.6 ± 6.8 (mg/dL) Brain Cr Levels 18.5 ± 2.0   15.3 ±1.7  20.3 ± 1.8  21.6 ± 1.7 (ng/g) Cortex GLUT-1 100 ± 3.3 130.4 ± 9.3122.8 ± 1.3  99.9 ± 7.3 Expression (% Control) Cerebellum GLUT-1 100 ±4.7 143.3 ± 8.0  75.6 ± 7.6  83.0 ± 4.2 Expression (% Control)Hippocampus GLUT-1 100 ± 6.3 105.3 ± 5.3 102.3 ± 5.5 103.7 ± 2.8Expression (% Control) Cortex GLUT-3 100 ± 8.0  387.9 ± 20.9  319.5 ±14.6  287.5 ± 31.3 Expression (% Control) Cerebellum GLUT-3 100 ± 7.3235.7 ± 1.1 206.3 ± 6.4 166.9 ± 2.2 Expression (% Control) HippocampusGLUT-3  100 ± 15.1 261.2 ± 3.0 198.6 ± 8.9 177.8 ± 4.7 Expression (%Control) Hippocampus Nrf2 100 ± 5.9  18.0 ± 2.9  38.9 ± 3.6  61.8 ± 1.4Expression (% Control) Hippocampus GFAP 100 ± 7.4 294.1 ± 7.0 251.2 ±9.1 204.9 ± 4.9 Expression (% Control) Hippocampus HNE  100 ± 18.0 342.2 ± 14.2  162.5 ± 12.4  140.1 ± 12.0 Expression (% Control)

The data above demonstrate that chromium pre-treatment can significantlyalleviate the negative side effects caused by hypoglycemia.

Example 2 Chromium and Insulin can form a Chromium-Insulin Complex

In order to evaluate chromium's potential to form a complex with insulinmolecules the following was performed.

100 μl of insulin (10 mg/ml) was mixed with 200 μl of chromiumhistidinate (“Cr-His”) (26 mg/ml) at room temperature (20° C.) whichformed a white precipitate. The precipitate was collected bycentrifugation and washed once with deionized water. The precipitate wasthen redissolved in 25 mM potassium phosphate buffer pH 7.4. Theoriginal supernatant and redissolved precipitate were then run through asize-exclusion column (for example, a 3 μM 100 Å column available fromAgilent Bio having a resolving range of 100-100,000 Da; larger moleculeseluting first) and analyzed with UV-Vis and ICPMS analysis.

Cr-His and insulin controls (i.e. same concentrations as in the Cr-Hisand insulin mixture) were run through the column. FIG. 14 shows the UV280 nm plot of chromium histidinate alone while FIG. 15 shows the UV 214nm plot of insulin alone.

FIG. 16 shows the UV 280 nm plot of the supernatant and FIG. 17 showsthe ICPMS plot targeting ⁵²Cr. Thus, in comparison to the Cr-Hiscontrol, the analysis of the supernatant indicated that much Cr-Hisremained in the supernatant.

FIG. 18 shows the UV 214 nm plot for the redissolved precipitate. Asshown, a peak at about ten minutes was detected. FIG. 19 shows the ICPMSplot targeting ⁵²Cr and FIG. 20 shows the ICPMS plot targeting ⁵³Cr forthe redissolved precipitate. Again a peak around ten minutes indicatedthe presence of chromium. The molecular weight of this elution at aroundten minutes was estimated at about 36 kDa. These data strongly suggestthat chromium forms a complex with insulin.

Example 3 Chromium-Insulin Compositions Raise Serum Insulin Levels andReduce Serum Glucose Levels in Normal Mice to a Greater Extent thanInsulin or Zinc-Insulin Compositions

In order to evaluate the effect of chromium-insulin compositions onserum insulin levels and serum glucose levels in normal mice, thefollowing was performed.

C57BL/6 mice (five mice per study group) were injected with 0.5 U/kg ofbody weight i.p. of insulin in three different forms: insulin alone,zinc-insulin, and chromium-insulin.

Serum insulin levels were measured over time. The serum insulin levelswere determined by rat insulin enzyme-linked immunosorbent assay (ELISA)kit from Crystal Chem (Downers Grove, Ill.).

FIG. 21 shows the results. As shown in FIG. 21, the same amount ofinsulin provided in the form of a chromium-insulin composition had adifferent, beneficial pharmacokinetic profile than the same amount ofinsulin provided as insulin alone and zinc-insulin compositions. Ofnote, the early time points indicate that chromium insulin injectionsraised serum insulin higher than the other compositions. Serum insulinlevels remained higher for chromium insulin in the early time period. Assuch, the data show that the compositions provided herein provideimproved absorption of insulin.

In order to determine if the improved absorption data correlated withimproved therapeutic effects, serum glucose levels were measured overtime using the FreeStyle blood glucose monitoring system (TheraSense,Phoenix, Ariz.). The data are shown in FIG. 22. shows that insulinprovided as a chromium insulin composition lowered serum glucose levelsto a greater degree than the same amount of insulin provided as insulinalone, or a zinc-insulin composition. These data demonstrate theimproved therapeutic efficacy of insulin, when provided in a compositioncomprising chromium.

Example 4 Chromium-Insulin Compositions Raise Serum Insulin Levels andReduce Serum Glucose Levels in Diabetic Mice Faster than Insulin Aloneand Faster than Zinc-Insulin Compositions

In order to evaluate the effect of chromium-insulin compositions onserum insulin levels and serum glucose levels in diabetic mice, thefollowing was performed.

KKAy mice (5 mice per study group) were injected with 0.5 U/kg of bodyweight i.p. of insulin in three different forms: insulin alone,zinc-insulin, and chromium-insulin. Serum insulin levels were measuredover time.

Serum insulin levels were measured over time. The serum insulin levelswere determined by rat insulin enzyme-linked immunosorbent assay (ELISA)kit from Crystal Chem (Downers Grove, Ill.).

FIG. 23 shows the results. As shown in FIG. 23, chromium-insulin had adifferent pharmacokinetic profile than both insulin alone andzinc-insulin. Of note, the early time points indicate that chromiuminsulin injections raised serum insulin higher than the othercompositions. Serum insulin levels remained higher in animals receivinginsulin in the form of a chromium insulin composition in the early timeperiod. These data confirm the observed increase in absorption ofinsulin, when provided as a chromium insulin composition.

FIG. 24 shows the serum glucose levels measured over time as determinedusing the FreeStyle blood glucose monitoring system (TheraSense,Phoenix, Ariz.). As shown in FIG. 24, chromium insulin injectionslowered serum glucose levels below insulin alone.

Example 5 Chromium-Insulin Compositions Lower Glucose Levels to aGreater Extent than Zinc-Insulin Compositions

In order to evaluate the effect of chromium-insulin compositions incomparison to zinc-insulin compositions in a diabetic rat model, thefollowing was performed.

Four experimental groups, each containing seven Wistar rats were formedas follows:

-   -   1) Control: injected with saline;    -   2) Type 1: injected with 65 mg/kg i.p. of streptozotocin (“STZ”)        (to model type 1 diabetes);    -   3) +ZnIns: injected with 65 mg/kg i.p. of STZ and injected with        6.23 mcg of ZnO and 3 IU of insulin per 100 g of body weight;    -   4) +CrIns: injected with 65 mg/kg i.p. of STZ and injected with        47.7 mcg Cr-His and 3 IU of insulin per 100 g of body weight.

Serum Glucose levels were calculated using Glucose Oxidase Peroxidasemethods (GOD/POD Kits) one hour after treatment. FIG. 25 shows theresults in graphical form. As shown in FIG. 25, the +CrIns had lowerserum glucose levels than the +ZnIns group. These data confirm thatinsulin, when provided as a chromium insulin composition, exhibitsunexpected and favorable therapeutic effects in terms of lowering serumglucose levels, when compared to insulin alone, or compositionscomprising zinc and insulin.

Example 6 Chromium-Insulin Compositions Maintain Normal Body Weight to aGreater Extent than Zinc-Insulin Compositions in Diabetic Rats

In order to evaluate the effect of chromium-insulin compositions incomparison to zinc-insulin compositions in a diabetic rat model, thefollowing was performed.

Four experimental groups, each containing seven Wistar rats were formedas follows:

-   -   1) Control: injected with saline;    -   2) Type 1: injected with 40 mg/kg i.p. of STZ;    -   3) +ZnIns: injected with 40 mg/kg i.p. of STZ and injected with        0.5 IU of zinc-insulin;    -   4) +CrIns: injected with 40 mg/kg i.p. of STZ and injected with        0.8 IU.

The rats were injected daily for eight weeks. The rats initial averageand final average body weights are shown below in Table 2.

TABLE 2 Effect of Insulin-chelate Type on Body Weight in Type 1 InducedRats Effect of insulin-chelate type on body weight of type-1 diabetesinduced rats (n = 7 per group) Response variables² Groups¹ Initial BW, gFinal BW, g BW Change, % Control 195.57 ± 4.97 253.00 ± 11.93^(a) +30.13± 8.04^(a) STZ 195.86 ± 5.23 166.29 ± 10.33^(c) −14.62 ± 5.96^(c) STZ +Zn-Insulin 195.43 ± 4.35 183.83 ± 7.96^(bc)  −4.77 ± 3.92^(b) STZ +Cr-Insulin 195.71 ± 4.90 195.57 ± 3.83^(b)  +0.25 ± 2.97^(b) p < 1.000.0001 0.0001

The data above demonstrate that treatment with insulin provided as achromium insulin composition can significantly alleviate the negativeside effects, such as weight loss, caused by hypoglycemia. Thebeneficial effect of the chromium insulin complexes was significantlygreater than that observed with a composition comprising zinc andinsulin.

Example 7 Composition Comprising Chromium and Insulin Having SynergisticEffect in Treating Diabetes

A first, second, third, and fourth subject having similar weight, age,insulin sensitivity, and other characteristics are identified as havingdiabetes. The subjects each present one or more symptoms associated withdiabetes, such as a fasting serum glucose level over 126 mg/dL.

The first subject is parenterally administered a control salinesolution.

The second subject is parenterally administered a dosage X of chromiumbetween 25 and 2,000 μg.

The third subject is parenterally administered a dosage Y of insulinbetween 1 unit and 500 units.

The fourth subject is parenterally administered a composition comprisingthe dosage X of chromium between 25 and 2,000 μg and dosage Y of insulinbetween 1 unit and 500 units.

The subjects' fasting serum glucose levels are measured before and afteradministration of the chromium. After administration of the chromium,the first subject is observed to no change in serum glucose level. Thesecond subject is observed to have a reduced serum glucose level. Thethird subject is observed to have a reduced glucose level that is lowerthan that for the third subject, and has become hypoglycemic. The fourthsubject is observed to have a reduced serum glucose level lower thanthat of the first and second subject, but higher than that of the thirdsubject, and not hypoglycemic.

Example 8

In order to evaluate the efficacy of a parenterally administeredcomposition comprising chromium and insulin, diabetic animals wereparenterally administered a composition comprising chromium and insulin,and indicators of metabolic function, diabetic profile, and markers ofhypoglycemic brain damage were compared in animals with and withoutadministration of chromium. In addition, the administration of chromiumwas compared to the administration of zinc.

Forty-two Wistar rats were assigned to one of 6 experimental groups: 1)positive control: rats injected with saline 2) type-1 diabetes group:rats injected with streptozotocin (STZ, 65 mg/kg i.p.) to damage betacells (n=35). Diabetic rats were then administered with a) none, b) Znalone (6.23 μg, ZnO), c) Cr alone (47.7 μg, Cr-histidinate), d)Zn-insulin (6.23 μg, ZnO+3 IU Ins/100 g BW), or e) Cr-insulin (47.7 μg,Cr-histidinate+3 IU Ins/100 g BW), daily for 26 days (n=7 per subgroup).Body weights were measured at the beginning and end of the experiment.

Blood samples were collected on days -2 (beginning), 0 (induction), 4,6, 12, and 26 for blood biochemistry. At the end of the experiment, ratswere sacrificed for brain tissue GLUTs (1 and 3). Data were analyzedusing one-way ANOVA with LSD option for mean comparison. Body weight atthe beginning of the experiment was not different across the groups.However, diabetic rats at the end of the experiment lost body weight ascompared to the control rats. Diabetic rats treated with Zn-Ins andCr-Ins lost less body weight than untreated diabetic rats. Diabetesinduction was associated with decreased serum insulin and total protein,and CK (creatinine kinase) levels and increased serum glucose, urea,creatinine, and K levels as well as AST (aspartate aminotransferase),ALT (alanine aminotransferase), ALP (alkaline phosphatase) and LDH(lactate dehydrogenase) activities.

Efficacy of CrIns to restore metabolic profile was equivalent orsuperior to ZnIns. During the experiment, injecting CrIns was moreeffective to reduce elevated serum glucose level than injecting ZnIns.Brain GLUTs expressions were depressed by diabetes induction. CrInstreatment was superior to other treatment choices in terms ofalleviating cerebral GLUTs expressions. In conclusion, it appears thatCrIns is superior to ZnIns to suppress hyperglycemia through dual effectof Cr and exogenous insulin, probably resulting from potentiated insulinaction and internalized glucose.

The results of the study are shown in Tables 2-7 and FIGS. 7-13.

TABLE 3 Effect of insulin-chelate type on body weight of type-1 diabetesinduced rats (n = 7 per group). Response variables² Groups¹ Initial BW,g Final BW, g BW Change, % Control 195.57 ± 4.97 253.00 ± 11.93^(a)+30.13 ± 8.04^(a) STZ 195.86 ± 5.23 166.29 ± 10.33^(c) −14.62 ± 5.96^(c)STZ + Zn 195.43 ± 7.63 175.71 ± 7.67^(bc)  −9.71 ± 4.03^(bc) STZ + Cr195.43 ± 5.94 176.57 ± 12.27^(bc) −10.17 ± 3.94^(bc) STZ + Zn-Insulin195.43 ± 4.35 183.83 ± 7.96^(bc)  −4.77 ± 3.92^(b) STZ + Cr-Insulin195.71 ± 4.90 195.57 ± 3.83^(b)  +0.25 ± 2.97^(b) p < 1.00 0.0001 0.0001¹Rats in control group was injected with saline. STZ = streptozotocin(STZ, 40 mg/kg i.p.); STZ + Zn-Insulin = STZ + Zn-insulin (0.5 IU);STZ + Cr-Insulin = STZ + Cr-insulin (0.8 IU) daily for 26 days.²Different superscripts within columns differ (p < 0.05).

As shown in Table 2, compared to all other treatment groups includingZnIns, CrIns resulted in the lowest reduction in body weight. These datasuggest that CrIns is superior to ZnIns in treating diabetes.

TABLE 4 Effect of insulin-chelate type on glucose pattern of type-1diabetes induced rats. Groups^(1,2) STZ + STZ + Response STZ + STZ + Zn-Cr- variables³ Control STZ Zn Cr Insulin Insulin Basal level  95 ± 1.59After STZ 324 ± 15.4 administration Day 4 Before 60 473 457 403 374 462After 69 439 303 237 118 94 6 Before 77 392 350 330 388 371 After 71 441372 349 120 95 12 Before 70 451 421 340 400 372 After 70 446 412 349 138104 21 Before 61 438 380 368 419 376 After 70 452 329 303 148 107 PooledSEM 25.33 ANOVA Group 0.0001 Time relative 0.0001 to injection Group xtime 0.0001 relative to injection Day 0.36 Group x day 0.50 Timerelative 0.0003 to injection x day Group x time 0.19 relative toinjection x day ¹Rats in control group was injected with saline. STZ =streptozotocin (STZ, 40 mg/kg i.p.); STZ + Zn-Insulin = STZ + Zn-insulin(0.5 IU); STZ + Cr-Insulin = STZ + Cr-insulin (0.8 IU) daily for 26days. ²Different superscripts within rows differ (p < 0.05).

TABLE 5 Effect of insulin-chelate type on blood biochemistry of type-1diabetes induced rats at the end of the animal experimentationGroups^(1,2) Response STZ + Zn- STZ + Cr- variables³ Control STZ STZ +Zn STZ + Cr Insulin Insulin p Insulin, 36.0 ± 1.4^(a)  19.4 ± 1.5^(c)20.9 ± 0.9^(bc ) 22.0 ± 0.8^(bc) 23.0 ± 1.1^(b)  23.9 ± 1.1^(b) 0.0001μU/mL Glucose, 128 ± 7^(e)  478 ± 20^(a) 456 ± 13^(ab ) 423 ± 15^(bc)416 ± 10^(cd) 379 ± 6^(d)  0.0001 mg/dL AST, U/L 141 ± 8^(c)  343 ±17^(a) 328 ± 17^(a)  291 ± 24^(ab) 288 ± 30^(ab) 268 ± 16^(b) 0.0001ALT, U/L 87 ± 6^(d)  220 ± 11^(a) 143 ± 9^(b)  125 ± 12^(bc) 115 ±9^(c )  102 ± 3^(cd ) 0.0001 ALP, U/L 133 ± 5^(d)  550 ± 13^(a) 453 ±11^(b)  442 ± 13^(bc) 429 ± 17^(bc) 411 ± 8^(c)  0.0001 LDH, U/L 1465 ±146^(c)  2716 ± 99^(a)  2564 ± 85^(ab )  2367 ± 52^(b ) 2523 ± 78^(ab) 2329 ± 83^(b)  0.0001 CK, U/L 15958 ± 3256^(a)   6188 ± 1166^(b) 6872 ±1248^(b) 6745 ± 934^(b)  7989 ± 523^(b)  8352 ± 338^(b) 0.001 Total 6.77± 0.18^(a)  6.01 ± 0.24^(b) 5.91 ± 0.07^(b)  6.07 ± 0.12^(b)  6.33 ±0.17^(ab)  6.67 ± 0.19^(a) 0.002 protein, g/dL Albumin, 2.86 ± 0.08^(a)  2.77 ± 0.10^(ab) 2.59 ± 0.06^(b)  2.74 ± 0.06^(ab)  2.67 ± 0.08^(ab) 2.84 ± 0.06^(a) 0.09 g/dL Urea, mg/dL 31.1 ± 1.4^(d)  83.6 ± 3.9^(a)65.6 ± 1.0^(b)  62.1 ± 2.5^(bc) 61.3 ± 2.9^(bc) 57.9 ± 2.1^(c) 0.0001Creatinine, 0.55 ± 0.02^(d)  1.60 ± 0.08^(a) 1.05 ± 0.07^(b)  0.90±0.08^(bc)  0.85 ± 0.14^(bc)  0.78 ± 0.09^(cd) 0.0001 mg/dL Uric acid,4.23 ± 0.29^(c)  7.67 ± 0.34^(a)  6.49 ± 0.69^(ab)  6.21 ± 0.57^(b) 5.75 ± 0.33^(b)  5.59 ± 0.16^(b) 0.0002 mg/dL K, mmol/L 4.65 ± 0.10^(c) 5.98 ± 0.20^(a) 5.53 ± 0.09^(b)  5.57 ± 0.12^(b)  5.48 ± 0.08^(b)  5.29± 0.15^(b) 0.0001 ¹Rats in control group was injected with saline. STZ =streptozotocin (STZ, 40 mg/kg i.p.); STZ + Zn-Insulin = STZ + Zn-insulin(0.5 IU); STZ + Cr-Insulin = STZ + Cr-insulin (0.8 IU) daily for 26days. ²Different superscripts within rows differ (p < 0.05). ³AST =aspartate aminotransferase; ALT = alanine aminotransferase; ALP =alkaline phosphatase; LDH = lactate dehydrogenase; CK = creatininekinase

As shown in Table 4, compared to all other treatment groups includingZnIns, CrIns was most effective at returning levels of serum insulin,CK, total protein, albumin, serum glucose, AST, ALT, ALP, LDH, urea,creatinine, and uric acid to control levels.

TABLE 6 Effect of insulin-chelate type on cerebral glucose transporter(GLUT) expressions in type-1 diabetes induced rats. Response variables²Group¹ GLUT-1 GLUT-3 Control 100.00 ± 3.50^(a)  100.00 ± 4.33^(a)  STZ31.94 ± 1.42^(d) 25.56 ± 3.00^(d) STZ + Zn 46.82 ± 3.23^(c) 43.41 ±3.53^(c) STZ + Cr 53.81 ± 3.53^(c) 49.41 ± 2.39^(c) STZ + Zn-Insulin75.47 ± 3.94^(b) 81.54 ± 5.80^(b) STZ + Cr-Insulin 89.71 ± 4.62^(a)95.60 ± 3.52^(a) p < 0.0001 0.0001 ¹Rats in control group was injectedwith saline. STZ = streptozotocin (STZ, 40 mg/kg i.p.); STZ + Zn-Insulin= STZ + Zn-insulin (0.5 IU); STZ + Cr-Insulin = STZ + Cr-insulin (0.8IU) daily for 26 days. ²Different superscripts within columns differ (p< 0.05).

As shown in Table 5, compared to all other treatment groups includingZnIns, CrIns resulted in the highest levels of GLUT-1 and GLUT-3expression. These data suggest that CrIns is superior to ZnIns tosuppress hyperglycemia and treat diabetes.

TABLE 7 Effect of insulin-chelate type on serum and brain chemicals (n =7 per group). Response variables² Brain Brain Serum Cr Brain CrSerotonin Tryptophan Groups¹ (mcg/g) (ng/g) (mcg/g) (mcg/g) Control75.17 ± 2.54^(a) 24.77 ± 1.52^(a) 640 ± 4^(a) 9.19 ± 0.43^(a) STZ 33.38± 2.86^(d)  8.81 ± 0.70^(d) 484 ± 5^(e) 4.01 ± 0.23^(d) STZ + Zn 33.65 ±2.26^(d)  9.05 ± 0.56^(d) 492 ± 5^(e) 4.54 ± 0.23^(d) STZ + Cr 48.64 ±2.70^(c) 11.87 ± 0.89^(c) 512 ± 7^(d) 4.74 ± 0.36^(d) STZ + 38.75 ±3.26^(d) 12.14 ± 0.76^(c) 552 ± 6^(c) 5.98 ± 0.29^(c) Zn-Insulin STZ +65.00 ± 2.63^(b) 16.71 ± 0.62^(b) 590 ± 7^(b) 7.81 ± 0.35^(b) Cr-Insulinp < 0.0001 0.0001 0.0001 0.0001 ¹Rats in control group was injected withsaline. STZ = streptozotocin (STZ, 40 mg/kg i.p.); STZ + Zn-Insulin =STZ + Zn-insulin (0.5 IU); STZ + Cr-Insulin = STZ + Cr-insulin (0.8 IU)daily for 8 weeks. ²Different superscripts within columns differ (p <0.05).

As shown in Table 6, compared to all other treatment groups includingZnIns, CrIns resulted in the highest levels of serum chromium levels,brain chromium levels, brain serotonin levels, and brain tryptophanlevels. These data suggest that CrIns is superior to ZnIns to suppresshyperglycemia and treat diabetes.

TABLE 8 Effect of insulin-chelate type on bands (n = 7 per group).Response variables² Kidney Kidney Kidney Kidney Brain Brain Group¹ oct1oct2 nfk mrp2 nfk ins Cntrl. 100.00 ± 2.81^(a)  100.00 ± 3.00^(a) 100.00 ± 1.84^(c) 100.00 ± 2.58^(c) 100.00 ± 2.80^(c) 100.00 ± 5.41^(a) STZ 36.56 ± 2.84^(e) 35.03 ± 1.71^(c) 156.54 ± 9.31^(a) 175.38 ±4.33^(a) 221.68 ± 2.80^(a) 40.10 ± 3.03^(c) STZ + Zn 50.02 ± 2.05^(d)51.38 ± 1.17^(d) 120.31 ± 1.65^(b) 148.73 ± 2.74^(b) 150.59 ± 2.73^(b)44.08 ± 3.31^(c) STZ + Cr 50.90 ± 1.71^(d) 53.50 ± 1.13^(d) 118.86 ±1.47^(b) 140.21 ± 2.05^(b) 134.24 ± 4.10^(b) 47.89 ± 4.67^(c) STZ +Zn-Ins 74.79 ± 1.66^(c) 70.41 ± 3.49^(c)  90.41 ± 3.30^(c)  92.79 ±4.60^(c) 106.06 ± 9.04^(c) 78.68 ± 4.25^(b) STZ + Cr-Ins 86.28 ±1.72^(b) 81.87 ± 5.46^(b)  85.80 ± 8.87^(c)  89.10 ± 4.29^(c)  89.90 ±8.58^(c)  88.31 ± 2.71^(ab) 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001¹Rats in control group was injected with saline. STZ = streptozotocin(STZ, 40 mg/kg i.p.); STZ + Zn-Insulin = STZ + Zn-insulin (0.5 IU);STZ + Cr-Insulin = STZ + Cr-insulin (0.8 IU) daily for 8 weeks.²Different superscripts within columns differ (p < 0.05).

As shown in Table 7 and FIG. 7, administration of the CrIns combinationresulted in post-administration serum-glucose levels of approximately100 mg/dl, which is a desirable euglycemic level. Thepost-administration serum glucose levels for the ZnIns combinationvaried from approximately 120-150 mg/dl. These data suggest that CrInsis superior to ZnIns to suppress hyperglycemia and treat diabetes.

As shown in Table 7 and FIGS. 8 and 9, the CrIns combination resulted inthe highest percentages of kidney OCT-1 and OCT-2 (organic cationtransporters) as compared to control levels. OCTs are important for therenal homeostasis of a number of physiologically important endogenouscations, including monoamine neurotransmitters, agmatine, andprostaglandins. OCTs are also necessary for the renal clearance of abroad range of exogenous substrates, including toxins, xenobiotics, andcommonly used drugs (e.g., metformin and β-blockers). (Thomas et al(2004) JPET 311:456-466). These data suggest that CrIns may enhance theefficacy of some diabetes drugs, including for example, metformin.

As shown in Table 7 and FIG. 10, the CrIns combination resulted in thelowest percentage of kidney NFK (nuclear factor kappa B) as compared tocontrol levels. NFK is a protein transcription factor that is requiredfor maximal transcription of a wide array of pro-inflammatory moleculeswhich are thought to be important in the generation of acuteinflammation. (Christman et al. (2000) Brain Pathology 10:153-162). NFKactivation induced by long-lasting oxidative stress has been shown to beresponsible for neuronal damage and consequent promotion of cell death.(Aragano et al (2002) Endocrinology 143(9):3250-3258).

As shown in Table 7 and FIG. 11, the CrIns combination resulted in thelowest percentage of kidney MRP2 (multidrug resistance related protein2) as compared to control levels. MRP2 is an ATP-binding cassette (ABC)transporter that functions in the organic anion transport system.(Sekine et al. (2006) Am. J. Physiol Renal Physiol 290:F251-F261).

As shown in Table 7 and FIG. 12, the CrIns combination resulted in thelowest percentage of brain NFK as compared to control levels.

As shown in Table 7 and FIG. 13, the CrIns combination resulted in thehighest percentage of brain insulin levels as compared to controllevels. These data suggest that CrIns is superior to ZnIns to suppresshyperglycemia and treat diabetes.

Example 9

A subject is identified as having early stage Alzheimer's disease. Thesubject presents with one or more symptoms including memory changes thatdisrupt daily life, challenges in planning or solving problems,difficulty in completing familiar tasks, confusion with time or place,trouble understanding visual images and spatial relationships, newproblems with words in speaking or writing, misplacing things and losingthe ability to retrace steps, decreased or poor judgment, withdrawalfrom work or social activity, and changes in mood and personality.

The subject is administered a composition comprising between 50 μg and5000 μg chromium and between 1 unit and 500 units of insulin. Thecomposition is administered parenterally. The subject's condition, asassessed by one or more symptoms of the disease, does not worsen, orimproves, over time.

Example 10

A subject is identified as having Alzheimer's disease by a routinedementia screening test, such as a clock drawing test, a time and changetest, a sniff test, or the like, and/or shows symptoms of Alzheimer's asevidenced by a PET scan.

The subject is administered a composition comprising between 50 μg and5000 μg chromium and between 1 unit and 500 units of insulin. Thecomposition is administered parenterally. The subject's condition, asassessed by one or more symptoms of the disease, does not worsen orimproves over a period of five days.

Example 11

A subject is identified as having Parkinson's disease by conventionalmethods. The subject presents with one or more symptoms includingtremors, stiffness (or rigidity) of muscles, slowness of movement(bradykinesia) and loss of balance (postural dysfunction).

The subject is administered a composition comprising between 50 μg and5000 μg chromium and between 1 unit and 500 units of insulin. Thecomposition is administered parenterally. The subject's condition, asassessed by one or more symptoms of the disease, does not worsen, orimproves, over time.

Example 12

A subject is identified as having mild cognitive impairment. The subjectpresents one or more symptoms including memory complaints corroboratedby an informant, objective memory impairment for age and education,normal general cognitive function, intact activities of daily living,and the subject does not meet criteria for dementia

The subject is administered a composition comprising between 50 μg and5000 μg chromium and between 1 unit and 500 units of insulin. Thecomposition is administered parenterally. The subject's condition, asassessed by one or more symptoms of the disease, does not worsen, orimproves, over time.

Example 13

A subject presents with symptoms of ADHD, including inattention (e.g.failure to give close attention, difficulties in sustaining attention,difficulties in organizing tasks and activities and easily distracted byextraneous stimuli), hyperactivity (e.g. difficulties in remainingseated, excessive motor activity in inappropriate situations, thepatient acts as if “driven by a motor”), and impulsivity (e.g.difficulties in awaiting turn, answer questions before they have beencompleted and often interrupts or intrudes ongoing conversation).

The subject is administered a composition comprising between 50 μg and5000 μg chromium and between 1 unit and 500 units of insulin. Thecomposition is administered parenterally. The subject's condition, asassessed by one or more symptoms of the disease, does not worsen, orimproves, over time.

The foregoing description details certain embodiments of the invention.It will be appreciated, however, that no matter how detailed theforegoing appears in text, the invention can be practiced in many ways.As is also stated above, it should be noted that the use of particularterminology when describing certain features or aspects of the inventionshould not be taken to imply that the terminology is being re-definedherein to be restricted to including any specific characteristics of thefeatures or aspects of the invention with which that terminology isassociated. The scope of the invention should therefore be construed inaccordance with the appended claims and any equivalents thereof

What is claimed is:
 1. A sterile injectable formulation comprising: anamount of a chromium-insulin complex, the chromium-insulin complexhaving between 4 and 6 chromium ions per insulin hexamer; and at leastone excipient.
 2. The formulation of claim 1, wherein the amount of thechromium-insulin complex is an amount sufficient to stabilize serumglucose levels in a patient in need thereof
 3. The formulation of claim1, wherein the sterile injectable pharmaceutical formulation providesgreater therapeutic effect than an equivalent amount of injectedinsulin.
 4. The formulation of claim 1, wherein the sterile injectablepharmaceutical formulation provides greater therapeutic effect than anequivalent amount of injected zinc-insulin.
 5. The formulation of claim1, wherein the chromium-insulin complex is dissolved in a solution. 6.The formulation of claim 1, wherein the chromium-insulin complex issuspended in a solution.
 7. The formulation of claim 1, wherein thechromium-insulin complex has a molecular weight that is between about 30kDa and 40 kDa.
 8. A method for reducing serum glucose levels in asubject in need thereof comprising: providing an injectablechromium-insulin formulation; and administering the composition to thesubject, wherein the administration reduces serum glucose levels in thesubject to a great extent after one hour than an equivalent amount of aninjectable insulin formulation or injectable zinc-insulin formulation.9. The method of claim 8, further comprising identifying the patient inneed of reduced serum glucose levels.
 10. The method of claim 8, whereinthe injectable chromium-insulin formulation includes chromium-insulincomplexes, the chromium-insulin complexes having between 4 and 6chromium ions per insulin hexamer.
 11. A method for reducing serumglucose levels in a subject having diabetes comprising: providing aninjectable chromium-insulin formulation; and administering thecomposition to the subject having diabetes, wherein the administrationreduces serum glucose levels in the subject to a great extent afterthree hours than an equivalent amount of an injectable insulinformulation or injectable zinc-insulin formulation.
 12. The method ofclaim 11, further comprising identifying the subject having diabetes.13. The method of claim 11, wherein the injectable chromium-insulinformulation includes chromium-insulin complexes, the chromium-insulincomplexes having between 4 and 6 chromium ions per insulin hexamer. 14.A method for increasing serum insulin levels in a subject in needthereof comprising: providing an injectable chromium-insulinformulation; and administering the composition to a subject, wherein theadministration increases serum insulin levels in the subject to a greatextent after thirty minutes than an equivalent amount of an injectableinsulin formulation or injectable zinc-insulin formulation.
 15. Themethod of claim 14, further comprising identifying a patient in need ofincreased serum insulin levels.
 16. The method of claim 14, wherein theinjectable chromium-insulin formulation includes chromium-insulincomplexes, the chromium-insulin complexes having between 4 and 6chromium ions per insulin hexamer.
 17. A method for increasing seruminsulin levels in a subject having diabetes comprising: providing aninjectable chromium-insulin formulation; and administering thecomposition to the subject having diabetes, wherein the administrationincreases serum levels in the subject to a great extent after one hourthan an equivalent amount of an injectable insulin formulation orinjectable zinc-insulin formulation.
 18. The method of claim 17, furthercomprising identifying the patient having diabetes.
 19. The method ofclaim 17, wherein the injectable chromium-insulin formulation includeschromium-insulin complexes, the chromium-insulin complexes havingbetween 4 and 6 chromium ions per insulin hexamer.